Chronicle of a Death Foretold and the Stranger Essay Example for Free

Chronicle of a Death Foretold and the Stranger Essay Conflict Resulting From the Negative Effects of Rigid Societal Expectations in A Chronicle of A Death Foretold by Gabriel Garcia Marquez and The Stranger by Albert Camus Everyone has felt the pressure of societal expectations during their lifetime. The negative effects society brings on one’s life can lead to a feeling of rejection towards the people who do not conform to meet those standards. Gabriel Garcia Marquez, author of Chronicle of a Death Foretold, and Albert Camus, author of The Stranger, both construct the external moral conflict of society versus the protagonist in order to critique the way society fails to accept the moral values of the people who differ from the norm. In Chronicle of a Death Foretold, Gabriel Garcia Marquez emphasizes the central conflict of society versus the protagonist, based on beliefs and values, through the controversy leading up to the murder of Santiago Nasar, which is based on family honor and pre-marital sex. When Pedro and Pablo Vicario ask their sister, Angela Vicario, who had stolen her virginity, her response is described as â€Å"She only took the time necessary to say the name †¦ and she nailed it to the wall with her well-aimed dart, like a butterfly with no will whose sentence has already been written. ‘Santiago Nasar,’ she said†(47). Describing Angela’s response as â€Å"only took the time necessary† indicates that she is trying to put the blame on Santiago, making him a scapegoat, in order to protect the true man who took her virginity before marriage. In the Latin American society, where the setting of the novella takes place, it is not acceptable for a woman to have pre-marital sex due to the beliefs and morals of Catholics. Santiago is represented as the butterfly â€Å"whose sentence had already been written†. Even though there is no evidence Santiago took Angela’s virginity, it is the twin’s duty to protect their sister. Therefore, Pedro and Pablo Vicario set out to kill the man who had stolen their sister’s virginity, Santiago Nasar, in order to protect their family. The twins tell everyone in town about their plan to murder Santiago, but the people in the community doubt their intentions: â€Å"twenty-two people declared they had heard everything said, and they all coincided in the impression that the only reason the brothers had said it was so that someone would come over to hear them†(51). The people in the town who â€Å"[coincide] in the impression that the only reason the brothers had said it was so that someone would come over to hear them† shows how oblivious the townspeople act owards a societal member’s life. Society looks past at the fact that a murder is about to occur, but focuses on the purpose; family honor, which is highly respected. Since it is unacceptable for a female to have sex before marriage, Santiago is viewed in a negative light by society because he is accused of taking the virginity of an unmarried woman. The debate surrounding Santiago’s death highlights the conflict between society and the protagonist, where Santiago is a man who is accused of committing an act that society does not accept. Albert Camus, in The Stranger, constructs the protagonist, Monsieur Meursault, as a man who has absurdist morals and values, which society does not accept. Meursault has an intimate relationship with Marie Cardona, a former typist, but the connection does not go beyond a physical attraction; â€Å"she wanted to know if I loved her. I answered the same way I had the last time, that it didn’t mean anything but that I probably didn’t love her† (41). By Meursault stating â€Å"it didn’t mean anything† and â€Å"I probably didn’t love her† highlights his absurdist views on life; he believes life contains no purpose, thus he cannot love Marie because loving Marie would give life a purpose, which Meursault does not believe. He simply associates with Marie, because he likes being around her. There is no need for an emotional connection because the physical appearance of her is satisfying enough. Absurdism is not accepted in society therefore it does not accept Meursault. He is an outsider in a world he did not choose. Meursault is a man who does not show his emotions very well, but instead focuses on his physical need; â€Å"I explained to him, however, that my nature was such that my physical needs often got in the way of my feelings† (65). Meursault saying â€Å"that my physical needs often got in the way of my feelings† emphasizes the simplistic life he live—a life without meaning. Showing emotions would signify a life with purpose, which Meursault does not believe. The Algerian society, in which the novella takes place, does not accept the type of lifestyle Meursault lives and believes every life should be lived with a purpose. Society’s expectations clash with Meursault’s values because his beliefs and outlooks on life are not accepted by society, which sets up the external conflict between the two. In the external conflict between society and the protagonist, Garcia Marquez emphasizes the negative effects society has on a person who goes against the social norm by showing the biased opinions towards the Pedro and Pablo Vicario regarding Santiago’s murder. After brutally stabbing Santiago to death, the Vicario twins sprint to the church to inform the priest of their barbaric act; â€Å"Both were exhausted from the barbarous work of death, and their clothes and arms were soaked and their faces smeared with sweat and still living blood but the priest recalled the surrender as an act of great dignity†(49). The priest calling the murder â€Å"an act of great dignity† shows how, in the Latin American society, family honor is highly valued, no matter how far it is taken. Even though the twins have committed the worst crime imaginable, it is acceptable because it was done in order to protect their family. Pedro and Pablo Vicario are viewed as meeting expectations, as opposed to Santiago, who fails to follow them. When the crime was brought to court, the twins had already won before it even started; â€Å"The lawyer stood by the thesis of the homicide in legitimate defense of honor, which was upheld by the court in good faith, and the twins declared at the end of the trial that they would have done it again a thousand times over for the same reason† (48). The lawyer stating the homicide as a â€Å"legitimate defense of honor† verifies that society values family honor over a man’s life. The court case represents the conflict of society against Santiago and since he goes against society’s values, Santiago ends up dying, indicating society’s victory. The twins do not receive any severe punishment, because of their intentions to protect their family. Through Santiago’s death, Garcia Marquez stresses the harmful effects society can have on someone whose beliefs differ from societal standards. Camus constructs Meursault’s trial to critique society’s lack of acceptance towards the people who do not meet expectations. Throughout the court case, Meursault is quickly judged by the prosecutor due to his actions concerning his mother’s death, â€Å"He said the truth was that I didn’t have a soul and that nothing human, not one of my moral principles that governs men’s hearts, was within my reach† (101). The prosecutor bluntly stating that Meursault â€Å"didn’t have a soul† and is incapable of having â€Å"moral principles that governs men’s hearts† highlights how society does not understand Meursault’s morals and values, thus critiques his character and neglects him. Meursault is looked down upon because of how he acted on the day of Maman’s funeral. He does not express feelings towards his mother’s death because he is an absurdist and believes death is inevitable. Society believes that there is purpose to every societal member’s life, and since Meursault shares absurdist views, he is not accepted by society. To close his final argument against Meursault, the prosecutor states, â€Å" ‘I ask you for this man’s head†¦never as strongly as today have I felt this painful duty made easier, light, clearer by the certain knowledge of a sacred imperative and by the horror I feel when I look into a man’s face and all I see is a monster. † (102). The whole trial is based around Meursault’s character, and him being called â€Å"a monster† stresses the fact that society is unwilling to accept anyone who does not follow the expectations. Meursault does not share the same views that society wants, and as a result, he is rejected because of his moral values. By asking â€Å"for this m an’s head† the prosecutor shows how society neglects the people who share different views and therefore want them out of society completely. Camus uses the trial and Meursault’s crime to emphasize the external conflict of society versus the protagonist, Meursault, to demonstrate how society does not accept people who share different moral values. The negative effects society has on the people who do not meet expectations are emphasized through the external conflict, based of moral values, between the protagonist and society in the Chronicle of a Death Foretold by Gabriel Garcia Marquez and The Stranger by Albert Camus. Both novellas show the harmful consequence faced by the protagonist, who do not conform to societal expectations, which evidently resulted in death.

How has Portugal been affected by globalization

How has Portugal been affected by globalization Economic globalization is not a recent phenomenon, it is the continuous evolution of developments that have been in train for a considerable amount of time. One may define economic globalization as the process by which markets and production in different countries are becoming increasingly interdependent due to the dynamics of trade in goods and services and flows of capital and technology European Commission (1997) cited in Sloman (2006). In other words, it involves the use of the factors of production on a world scale. The aim this essay is to discuss the impact of globalization in the economy of Portugal. Firstly, this paper will examine the relation between the globalization concept and the Portuguese integration in the European Union, explain in what forms its has been beneficial and describe the job creation in the service sector brought about the development of new information, production technologies and the expansion of tourism. Secondly, it is going to discuss the different negative repercussions of globalization in the various sectors of economic activity, analyse the socio-economic consequences of the relocation of multinationals, consider certain foreign investments in Portugal and expose certain possible disadvantages of having a single currency. The European Union as Cardoso and Ferreira (2000) asserts is currently the most successful example of regional economic integration which reflects the present Era of globalization. Portugal as a member of it since 1986 and as consequence of the European integration process has been experiencing considerable political, social and mainly economic changes. According to the above mentioned author, the various Portuguese areas of economic activity have been profoundly influenced by the European regulations and policies. With the creation of a common European market and a single currency, several constraints that limited in some extent the efficiency of business organizations and the full employment of their resources have been suppressed, the Portuguese companies gained the opportunity to explore economies of scale and to specialize in certain good and services through comparative advantage, improved their position to negotiate internationally, eliminated the exchange rate uncertainty, re duced inflation rates and enhanced competition which stimulates greater economic efficiency. Moreover, Cardoso and Ferreira (2000) further affirms that the gains of greater economic integration and interdependence between countries due to the globalization of the economy through the European Union also involve savings in foreign exchanges and transport costs. Furthermore, according to Lima et al. (2006), the above mentioned regional economic integration with its respective technological development of telecommunications and transport also permitted for example the organizational and technological restructure of the banking sector and stimulated employment in the service sector, mainly in the tourism sector. Considering the case of tourism, as Cardoso and Ferreira (2000) explains, this sector that in Portugal assumes significant social, cultural and particularly economic importance in creating jobs, increasing income per inhabitant, in the development of skilled labour, economic dive rsification and infrastructures has been profoundly positively affected along with different various areas of economic activity by the globalization and the European regulations and policies. Currently the total contribution of Travel and Tourism to the Portuguese Gross Domestic Product, including its wider economic impacts, is forecast to rise by 2.4% from à ¢Ã¢â‚¬Å¡Ã‚ ¬25.7bn (14.7% of GDP) in 2011 to à ¢Ã¢â‚¬Å¡Ã‚ ¬32.6bn (16.2%) by 2021 (World Travel and Tourism Council, 2011) and one may deduce that the above mentioned economic facts are due to the present globalization that made the tourism industry grow mainly with the fusion of cultures, transports revolution and deregulation policies as Wahab and Cooper (2001) assert. In addition, Lima et al. (2006) corroborates the above mentioned arguments by pointing out the fact that jobs in the hotel and restaurant sector increased by 9.4% between 1998 and 2004 and it also asserts that there has been noteworthy investments in transpor t and telecommunications, 30,000 jobs were created between 1998 and 2004 in this sector and it represented in 2006 7.4% of the service jobs. Furthermore, as a positive impact of the globalization in the countrys economy, Portugal since its integration in the European Union has always benefited from various structural funds and programs to encourage economic growth, higher competitiveness and to help reduce disparities among regions. As an example, one may consider the Portuguese archipelago island of Madeira which according to Beirman (2003) has been apportioned since 1986 structural funds to develop and modernize the region that made possible the construction of numerous infrastructures as roads, bridges, schools, airports, ports and health clinics. On the other hand, globalization also affected negatively the economy of Portugal in various aspects. According to Lima et al. (2006), the liberalization of the global economic markets encouraged the relocation of national and foreign industries from Portugal to other countries with more profitable production costs, which ultimately resulted in increasing unemployment in certain economic sectors, particularly in the automobile, footwear and textile sectors. As Lima et al. (2006) explains, the effects of the above mentioned aspects are considerably different in the various economic activities. In the case of industry, according to Lima et al. (2006), the number of jobs has been cut significantly by 105,000 jobs between the year 2000 and 2006. In contrast, over the same period, approximately 417,000 new jobs were created in the service sector. However, the above mentioned author further notes that precarious work situations are more common in the service sector. On one hand, from the a nalysis of the above mentioned arguments one may notice that globalization in Portugal creates in some situations new jobs and opportunities to expand companies, but on the other, provokes significant qualified and unqualified unemployment. According to the European Commission cited in Lima et al. (2006), the sluggish European economic growth combined with the acceleration of the liberalization of World Trade resulted in a loss of 860,000 textiles and clothing jobs in EU between 1998 and 2010; sectors that are considerably important for the Portuguese economy. The footwear and textile industries are the source of a considerable amount of jobs in Portugal and they have been severely affected by the relocation of enterprises to countries that offer fewer bureaucracies, less strict labour criteria, financial benefits and cheaper factors of production. According to the Portuguese national statistics institute cited in Lima et al. (2006), Portugal lost 90.000 jobs between 1998 and 2004, reducing its industrial employment from 23.5% in 1998 to 19.4% in 2004. In addition, at the above mentioned period, as Lima et al. (2006) asserts, Chinese entrepreneurs have been investing in the textile and clothing sector, a phenomenon that has previously occurred in Italy where the Chinese business management method, based on production at considerable low margin of profit and high volumes of sales has raised several issues regarding unfair competition. Although one may initially suppose that the above mentioned foreign investment is beneficial to the economy of the country, in reality it may be perhaps considerably prejudicial in several aspects since local small and medium size industries possess difficulty in competing with the Chinese production costs. Regarding other industries, particularly the automobile industry, according to Lima et al. (2006), has also been affected since 1990 by industrial relocation for mainly the above mentioned reasons; There was a 9% drop in jobs i n this sector between 1998 and 2004 (ibid). As the above mentioned author further notes, the closing of the Opel factory in Azambuja in 2006 left 1.200 people without a job and Renault closed its Setubal factory and invested in various new factories in Brazil and Slovenia. Consequently, unemployment as Lipsey and Chrystal (2007) point out, involves numerous micro and macroeconomic issues that damage the economy of Portugal, including the loss of the individuals income, a decline in their living standards, social deprivation, negative multiplier effects, a loss of potential national output, a waste of resources (labour) and fiscal costs since the government loses tax revenues and possesses the necessity of spending more on welfare payments for unemployed family members. Moreover, as Sloman (2006) make clear, due to the current global and regional interdependence Portugal is affected by the economic health of other countries and by their governments policies, issues in any area of the world can significantly affect Portugal through trade and financial markets, despite the eventual geographical distance. The present Portuguese economic situation reflects the vulnerability of the country to financial crisis. Finally, the European Monetary Union that is one of the examples of todays globalization, may be prejudicial to the Portuguese economy if Portugal possesses for example higher rates of inflation and if consequently its national enterprises possess difficulty in competing with the rest of the European Union. With a separate currency Portugal could allow its currency to depreciate and prevent being a depressed region of Europe with rising unemployment. In conclusion it seems apparent that the economy of Portugal has been profoundly affected by globalization. This essay has shown that it is possible to identify in Portugal numerous positive aspects and a significant amount of negative consequences of the global and regional increasing interdependence. One may deduce that the current concerning economic Portuguese situation is in part a reflection of the international financial crisis and the countrys vulnerability to the exterior. Nevertheless, from the analysis of the above mentioned arguments and despite the referred serious prejudicial implications of globalization, it seems undoubtable that globalization in recent years has contributed to the economic prosperity of Portugal. Hence, this essay suggests to minimize the negative effects of globalization in the Portuguese economy, the investment in education to qualify the work force and gain competitiveness, creation of more jobs in viable sectors as tourism, possible greater monit oring of financial institutions to prevent unexpected issues and increased international co-operation to solve the current and future economic problems.

Coca-Cola SWOT Analysis :: Business Management swot Analysis

Coca-Cola SWOT Analysis SWOT stands for Strengths Weakness Opportunities Threats. SWOT analysis is a technique much used in many general management as well as marketing scenarios. SWOT consists of examining the current activities of the organisation- its Strengths and Weakness- and then using this and external research data to set out the Opportunities and Threats that exist. Strengths: Coca-Cola has been a complex part of world culture for a very long time. The product's image is loaded with over-romanticizing, and this is an image many people have taken deeply to heart. The Coca-Cola image is displayed on T-shirts, hats, and collectible memorabilia. This extremely recognizable branding is one of Coca-Cola's greatest strengths. "Enjoyed more than 685 million times a day around the world Coca-Cola stands as a simple, yet powerful symbol of quality and enjoyment" (Allen, 1995). Additionally, Coca-Cola's bottling system is one of their greatest strengths. It allows them to conduct business on a global scale while at the same time maintain a local approach. The bottling companies are locally owned and operated by independent business people who are authorized to sell products of the Coca-Cola Company. Because Coke does not have outright ownership of its bottling network, its main source of revenue is the sale of concentrate to its bottlers. Weaknesses: Weaknesses for any business need to be both minimised and monitored in order to effectively achieve productivity and efficiency in their business’s activities, Coke is no exception. Although domestic business as well as many international markets are thriving (volumes in Latin America were up 12%), Coca-Cola has recently reported some "declines in unit case volumes in Indonesia and Thailand due to reduced consumer purchasing power." According to an article in Fortune magazine, "In Japan, unit case sales fell 3% in the second quarter [of 1998]...scary because while Japan generates around 5% of worldwide volume, it contributes three times as much to profits. Latin America, Southeast Asia, and Japan account for about 35% of Coke's volume and none of these markets are performing to expectation. Coca-Cola on the other side has effects on the teeth which is an issue for health care. It also has got sugar by which continuous drinking of Coca-Cola may cause health problems. Being addicted to Coca-Cola also is a health problem, because drinking of Coca-Cola daily has an effect on your body after few years. Opportunities: Brand recognition is the significant factor affecting Coke's competitive position.

Constructing Fantasy in Hitchcocks Vertigo Essay -- Alfred Hitchcock

Constructing Fantasy in Hitchcock's Vertigo The amount of critical analysis surrounding Alfred Hitchcock's Vertigo is itself dizzying, but as the film has recently been restored, it seems appropriate to provide it with a fresh critical reading. The purpose of this paper then, is to draw this film out of the past with a reading that offers not only a new way of understanding it, but a close look at the culture that produced it. Specifically, Vertigo offers its most exciting ideas when contextualized in a culture of consumerism. Consumerism shaped the film, and also shapes the way we view it. The desire of the consumer is the driving force behind not only our economy, but our mode of seeing the world, and seeing films. As consumers, we are always looking for, and looking at, new commodities, especially clothing. We gaze at clothing in shop windows. We purchase it and wear it, making it visible to others. Indeed, the desire to buy clothing is linked closely to our desire to show it off. We shop in a visual economy, a visual culture of consumption. To understand this culture it is important to understand the historical figure of the flà ¢neur. The flà ¢neur is a wandering male consumer of images who is, and was, particularly in the nineteenth century, the visual and economic agent at the center of consumer culture. He is also at the center of Vertigo, personified in the main character, Scotty. The flà ¢neur is an inveterate urban wanderer and voyeur who is at home in the public spaces. In the words of Baudelaire, "for the perfect flà ¢neur, for the passionate spectator, it is an immense joy to set up house in the heart of the multitude, amid the ebb and flow of movement" (qtd. in Brand 5). Walter Benjamin, in his work on the... ...lso of women displayed in windows. 3 Sometimes coincidence aids criticism. Kim Novak was, according to Hitchcock, quite proud of the fact that she didn't wear a bra during the filming of Vertigo (Truffaut 248). Works Cited Brand, Dana. The Sectator and the City in Nineteenth-Century American Literature. Cambridge: Cambridge UP, 1991. Gleber, Anke. The Art of Taking a Walk: Flanerie, litera ture, and Film in Weimar Culture. Princeton: Princeton UP, 1999. Friedberg, Anne. Window Shopping: Cinema and the Postmodern. Berkeley: U of California P, 1993. Simmons, Patricia. "Women in Frames: The Gaze, the Eye, the Profile in Renaissance Portraiture." The Expanding Discourse. Ed. Norma Broude and Mary Garrard. New York: Harper Collins. 39-57. Steele, Richard. "Spectator No. 454" 1712. The Spectator, A new edition. Cincinnati: Applegate & Co., 1857.

Persuasive research paper Essay

Everyday as we commute down the road we see motorcyclist drive past us. What is the one thing that we can all agree individuals riding motorcycles have in common? It is not a trick question. The answer is very simple; they all share the commonality of riding a motorcycle. What is in fact is very distinct however, are the choices of attire when operating their motorcycle. Some individuals are brave enough to wear shorts, tank tops, and sandals. On the opposite end of the spectrum, you have some of the wiser ones that chose to wear a helmet, gloves, protective jacket, eye protection etc. Why the distinct difference? The fact is that a great percentage of riders refuse to wear the proper protective equipment. Due to an increase in motorcycle riders within the recent years, a national protocol requiring certain equipment, such as a helmet, to be worn when riding a motorcycle should be instituted. There are many contributing factors to motorcycle fatalities, however there can be a culture of change, specially with the proper knowledge on how each piece of safety equipment can help at preventing injury or death Every year that passes by, notice that more and more motorcycle share the road with our  automobile drivers. We might wonder why there has been a shift in choice of transportation. Is this a trend or fad that the population is going through? According to the Governors Highway Safety Association, â€Å"National data from 1976 to 2012 suggest that motorcyclist fatalities track motorcycle registrations quite closely and that registrations track inflation-adjusted gasoline prices. If the economy continues to improve and gasoline prices remain high, then motorcycle 1 Tenorio registrations, travel, and fatalities will continue to rise unless active measures are taken†. (Hedlund). Not only do we think there are more motorcycle riders on the road, they have proven this to be true. Motorcycles are generally more fuel-efficient than cars, making them a very good alternative mode of transportation when gas prices stay at a consistent high price. It is basic mathematics; if there are more motorcycles there is more individuals susceptible to accidents. Additionally, they have proven that motorcycles are more apt to be involved in a motor vehicle accident than any other vehicle. Data collected in 2007 proved that per vehicle mile driven,motorcyclist were approximately 37 times more apt to die in a motor vehicle accident and nine times more probable to be injured in an accident. They also researched the ability of a helmet to protect against fatal injuries in motorcycle accidents. NHTSA estimates that helmets saved the lives of 1,829 motorcyclists in 2008. If all motorcyclists had worn helmets, an additional 823 lives could have been saved. (Motorcycles: Traffic Safety Facts – 2008 Data). As motorcycles become more abundant, it is imperative that we reduce the probability of death as much as  possible. As proven above, helmet wear can be a life or death-determining factor. You can force motorcycle operators to wear helmets by implementing laws, but the combination of alcohol and motorcycle operation can have a devastating impact despite helmet wear. When operating a motorcycle an operator needs all of their senses at full capacity. Alcohol is central nervous system suppressant substance, causing you body to have a reduced reaction time when the situation arises. The reported helmet use rate for motorcycle riders with BAC levels higher than  the legal limit killed in traffic crashes was 46 percent, compared with 66 percent for those with no alcohol (Motorcycles: Traffic Safety Facts – 2008 Data). Not only does alcohol reduce reaction times, it also has an impact on your ability to make rational decisions. It makes individuals push the limits of their motorcycle and their riding ability to levels they normally 2 Tenorio would not, and the majority of the time while not wearing the equipment they should. In 2011, the NHTSA calculated 4,323 motorcyclists were killed, and 33% (1426) of the riders were under  the influence of alcohol (Watson). How can the country as a whole help reduce the amount of fatalities we currently have due to motorcycle accidents? It is not a very simple answer. It would require involvement from both the people and the government to make this happen. One way the government can aid in the reduction of motorcycle fatalities is through the implementation of regulations, which require and enforce the wear or motorcycle protective equipment. Dating back to 1966 the government tried to impose the requirement of helmet wear by the states. They tried to do this by threatening with the reduction of federal-aid highway construction funds for the states that did not comply with the implementation of universal helmet use law by 1967. By 1975 all but 3 states had adopted and implemented such laws. Unfortunately the Supreme Court deemed this law unconstitutional. Shortly after revoking the Act, states gradually began to weaken helmet wear laws, since it was no longer a federal requirement (Helmet Laws). Much like seat belt laws have been implemented across the majority of the states due to increased survivability rate when  involved in an accident, the wear of helmets when operating a motorcycle should be mandated. The responsibility should not only be weighted only on the federal and state governments, individuals should take responsibility also. Many non-profit organizations work diligently to tray and raise motorcycle safety awareness with thinks like bumper stickers, fund-raising rides, and bike meets. Additionally insurance companies have aided in the increased awareness by handing out information pamphlets at locations like Bike Week in Daytona Beach. Another factor that  aids in the reduction of motorcycle fatalities is proper operation education. Florida is one of many states that require the operator to take a Motorcycle Basic Riders course in order to be able 3 Tenorio to receive the motorcycle endorsement on their licenses. Without this endorsement you cannot legally operate a motorcycle. With this course even people that have never been on a motorcycle can learn the basic in order to operate it on the roads. The Motorcycle Safety Foundation (MSF) offers motorcycle rider education and training programs and courses, and supports governmental  programs by participating in research and public awareness campaigns and providing technical assistance to state training and licensing programs (Morris). The Department of Defense, more specifically the United States Air Force, uses courses from the Motorcycle Safety Foundation to teach the military riders how to operate a motorcycle. In order for an individual to operate a motorcycle they have to complete the basic riders course. Within one year of the completion of the initial course they are required to complete an intermediate course such as the basic riders  course 2, advanced riders course, or the sport bikes handling course. Once these two requirements are complete, they are required to do refresher training every five years. In addition to the training, the department of defense requires all members, military and civilian, to wear protective equipment while driving on any DOD installation. That protective equipment consists of: helmet, gloves, durable over the ankle footwear, long sleeve shirt or jacket, long durable pants, and eye protection. If not properly equipped, individuals are not allowed to enter the installation. If the DOD is doing this to help keep the members of the military community safe, why shouldn’t the rest of the country follow in those footsteps? When we think motorcycle safety, 90 percent of the time the first image that comes to mind is a helmet, as it should. The helmet is the single-handedly the most important piece of safety equipment that a motorcycle rider shouldn’t go without. However, there are many other rider protective equipment components that play a vital role in the safety of the person. Between 2001 and 2008, more than 34,000 motorcyclists were killed and an estimated 1,222,000 persons. 4 Tenorio were treated in a U. S. emergency department for a non-fatal motorcycle-related injury (Motorcycle Crash-Related Data). This data supports the thought process that even though helmets are crucial at protecting against head injuries, there are many other portions of the body that are at harms way if not properly covered. 75 percent of the non-fatal emergency room visits involved parts other than the head. The other attire that might contribute to a safer ride includes, but not limited to, long durable pants, durable top, gloves, durable over-the-ankle footwear, and reflective equipment. Despite that it will probably never be deemed mandatory to wear these items, it is important for riders everywhere to understand the devastating effects an accident can have on their bodies when choosing not to wear the proper gear. There is a common misconception that the gear makes the ride more uncomfortable and, it is believed that it makes it more difficult to operate and maneuver the motorcycle. That is a myth! Properly fitted helmets of decent quality not only will it protect your head, but also a full-faced helmet will make for a more comfortable ride. The helmet does this by preventing foreign objects and debris from  constantly hitting the riders face, and most importantly from landing in the eye. Gloves that fit snug the hand will protect it from road rash in the event that you make contact with the pavement and it also improves handgrip with the handlebars aiding with better handling. There are gloves out on the market that have additional padding in the palm of the hand, to help with comfort and provide support and a barrier in the event of a fall. The same concept can be applied to footwear. It is unbelievable that there are people out there that would ride a bike in flip-flops and think it is comfortable. Not only does it not protect the appendages, but also it makes it harder to control the bike. When choosing footwear you have to find a medium between protection and comfort. Wear something that provides the proper amount of protection but does not hinder your ability to control or maneuver the motorcycle. 5 Tenorio Choosing comfort over safety should never be an option. More specifically when you are talking about the portion of your body that controls all bodily functions. With the implementation and enforcement of a universal helmet law, the fatality rate of motorcycle accidents would  decrease. In the past the universal helmet law failed. With that in mind, we can learn from our mistakes and see trough an effective and legal legislation. The ultimate goal is not to interfere with he rights of individuals, but to help protect the citizens so they can continue to enjoy the freedoms we have in the United States. The amount of information revolving around motorcycle safety out for public access is almost overwhelming. Therefore, there shouldn’t be an excuse why people refuse to wear gear that will only help protect them and their bodies from the dangers of riding a motorcycle. Works Cited 6 Tenorio Hedlund, James. â€Å"Spotlight on Highway Safety. † Motorcyclist Traffic Fatalities by State: 2012 Preliminary Data. Governors Highway Safety Association, 1 Apr. 2013. Web. 09 July 2014. â€Å"Helmet Laws. † State Motorcycle and Bicycle. Governors Highway Safety Association, 1 July 2014. Web. 06 July 2014. Morris, C. C. , Ph. D. â€Å"Motorcycle Trends in the United States | Bureau of Transportation Statistics. † Motorcycle Trends in the United States | Bureau of Transportation Statistics. Bureau If Transportation Statistics, 14 May 2009. Web. 07 July 2014. â€Å"Motorcycle Crash-Related Data. † Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 14 June 2012. Web. 06 July 2014. National Highway Traffic Safety Administration. Motorcycles: Traffic Safety Facts – 2008 Data (2008): 1-6. National Highway Traffic Safety Administration. NHTSA’s National Center for Statistics and Analysis, 1 Dec. 2008. Web. 22 June 2014. Watson, Tim. â€Å"What The Latest NHTSA Fatality Stats Reveal About Motorcycle Safety. † Ride Apart RSS2. Ride Apart, 29 May 2013. Web. 09 July 2014. Workman, Danny. â€Å"Deadly Motorcycle Accident Statistics. † Examiner. com. The Examiner, 28 May 2009. Web. 09 July 2014. 7.

GATTACA: ‘Vincent is supposed to be weak. Yet his strength of character is the key to the story.’ Essay

‘Vincent is supposed to be weak. Yet his strength of character is the key to the story.’ Discuss. The film text ‘Gattaca’, directed by Andrew Niccol can be seen as a piece that draws many parallels to the world that we live in today. One such parallel is the fact that often in society, the ones who are at a disadvantage are the ones who display the greatest strength of character. Niccol uses Vincent as the vehicle through which he demonstrates how strength of character can neutralise and overcome disadvantages faced. Gattaca portrays a society that no longer takes into consideration a person’s character. Vincent’s refusal to ‘play the hand he was dealt’ is his key to success. The text makes a clear distinction between ‘human’ and genetic traits. So clear is the distinction that Niccol uses separate vehicles to display each set of traits. As the traits and the vehicles clash, Niccol creates a sense of sympathy for Vincent as he is portrayed as the disadvantaged and weak in society in the sense that he does not possess the physical and mental assets that make him a valued member of society. A key example of this is the recurring games of ‘chicken’ between Vincent and his genetically superior brother Anton. Anton beats Vincent time and time again as is expected of Anton by society. However, when Vincent bests his brother one day an emphasis is placed on the manner in which he beat his brother. â€Å"You wanna know how I did it? I never saved anything for the swim back!† This creates the impression to the viewer that Vincent did not win with superior stamina, skill or physical strength, rather he won as he possessed traits lik e courage, determination and grit, traits that are lost in the geno-centric society in which he loves but traits that are valued and admired in todays society. The text can be seen as parallel to today’s society as often it is those who are faced with the greatest disadvantage who succeed. Vincent’s journey is a classic tale of 1 against the world or in this case 2 against the world. At every bend society is there to stop Vincent from achieving his lifelong dream of going to space. Even his parents try to prevent him from attaining  his goal, â€Å"Vincent, the only way you’ll see the inside of a spaceship is if you’re cleaning it.† This coming from his father can be seen as the ultimate discouragement but instead Vincent uses it to spur himself onwards and upwards both literally and figuratively. In relation to the real world, notable figures like Oscar ‘Blade Runner’ Pistorious overcame what is seen as a severe athletic disability in today’s society to compete at the 2012 London Olympics. While it is not uncommon to hear of ‘rags to riches’ tales in todays society, the one notable difference between today’s society and Vincent’s society, is that disadvantaged people are given every chance to make the most of their life (in 1st world countries), yet in Vincent’s society the disadvantaged are shunned and are viewed as a blight on society. Gattaca is a reminder that you cannot judge one on their outwardly appearance or in Gattaca’s case, a persons genetic composition. As the saying goes, ‘don’t judge a book by its cover.’ Vincent is once again used as the vehicle through which Niccol conveys this message. To the eyes of society, Vincent is an ‘invalid’ with inferior physical capabilities, high risk of heart failure and other deficiencies and with a â€Å"life expectancy of 30.2 years.† However, because of the ‘faceless’ society that Vincent lives in (which refers to personality as much as it does to physical appearance) he lacks the opportunity to progress with his life as his strengths of character are not held in anywhere near the same esteem as desirable genetics are. Vincent claims, â€Å"There is no gene for fate.† This is in reference to the fact that he is of the belief that genes aren’t everything, even if society has such beliefs. Rather there is an element in every human being that makes him or her truly unique and this is what Vincent prides himself on, the fact that his assets which are undeterminable by genetics are what makes him the person that he his. While the society in which Vincent lives in regards Vincent as weak and inferior, his character is both key to his success and the story as without his steely resolve and fierce determination there would be no story as society would continue to function as it was designed. Parallels can be drawn with Vincent’s plight and the journey’s of figures in our society like Oscar Pistorious. Gattaca serves to compound the message that has been  passed down through generations by reasserting that you shouldn’t judge a person before you know them personally. Vincent refuses to ‘play the hand he was dealt’ and this is the focal point around which Gattaca was told.

Lowering The National Drinking Age - 1698 Words

Lowering the National Drinking Age Winston Churchill was infamous for his one liners and occasional drunken outbursts. One night at a party, he shocked a rather prominent woman with his drunken atrocities. Insulted, she turned to him and said, â€Å"Mr. Churchill, you are as drunk as a dog.† The Prime Minister returned, â€Å"Madam, I may be very drunk, but you are very ugly. But tomorrow,† he added, â€Å"I shall be sober† (Churchill, W). The use and abuse of alcohol is a centuries old vice that has circumnavigated the globe and all eras of humanity; young and old, man and woman. Alcohol is an inhibitor of logical thought. However, it is an enjoyable pastime as well as custom in almost every society as long as it is used within reason. Recently however, laws have been placed in many nations restricting drinking to only certain age groups. In America, the legal age to purchase and consume alcohol has fluxuated between 18 and 21, coming to rest at 21 in 1984. (CIT E) This law is understandably strict, but also somewhat toying with a person s free abilities. Aggravating the threat of binge-drinking and alcohol poisoning, this exceptionally high age limit has promoted more hindrances than benefits. Currently a great debate among lawmakers is whether the minimum legal drinking age (MLDA) should be lowered to 18; mirroring other nations in their practices. A lower limit would be exceptionally beneficial and fair legally, health-wise, and economically towards not only young adults, but societyShow MoreRelatedAccording To â€Å"College Drinking,† Almost Two Out Of Three1388 Words   |  6 PagesAccording to â€Å"College Drinking,† almost two out of three college students engage in binge drinking. Binge drinking is a pattern of drinking that brings blood alcohol concentration (BAC) levels to 0.08 g/dL or higher (â€Å"College Drinking†). 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This seems to be a problem all over theRead More18 vs. 21: Drinking Age1389 Words   |  6 Pagesdo people only want to change the drinking age from 21 to 18, when there are other activities that have limit of age such as marriage at 18, driving at 16 and 35 to be a president? Alcohol plays a major role in today society, which becomes a controversial issue among teens. Alcohol is a mind-altering chemical that is potentially more dangerous than any other drug and can be very destructive. For past few years, many people are trying to lower the drinking age without knowing the negative eff ectsRead MoreChallenging The Legal Drinking Age1689 Words   |  7 PagesEnglish Language 25 July 2014 Challenging the Legal Drinking Age The Minimum Legal Drinking Age (MLDA) has been challenged since the passing of the National Minimum Drinking Age Act of 1984 that raised the drinking age to twenty-one in all fifty states (Ogilvie). 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Handling, storage and disposal of samples - Free Essay Example

Sample details Pages: 33 Words: 9774 Downloads: 2 Date added: 2017/06/26 Category Statistics Essay Did you like this example? Expectations of a Health Care Professional Handling, Storage and Disposal of Samples In the histology laboratory all specimens arrive fixed in 10% buffered formalin. In the laboratory, the specimen and the request form are labeled with the same lab number. The specimens are left in the same order the lab number is given and processed. Safety gloves and an apron are worn when processing the specimen. Unfixed specimens received in a plane container are fixed in 10% formalin which is commercially prepared and left for one day to process. This is done if the specimen requires fixation. Certain specimens are an exception to this rule. Lymph nodes are wrapped in gauze when lymphoma is suspected, skin sections for Immunofluorescence due to Pemphigus vulgaris are suspended in saline solution, and frozen sections are not fixed since fresh tissue is sectioned for microscopic examination. Don’t waste time! Our writers will create an original "Handling, storage and disposal of samples" essay for you Create order Whether the result has been reported or not determines which samples are disposed and stored if the result has been reported or not: After the samples are processed the pieces of the sample that were not inserted in the cassette are placed back inside the respective container. The fixed specimen in the container is refrigerated until the result is reported (figure1). 3 weeks after reporting the result a disposal list is created and the specimens to be disposed are packed inside boxes, labeled and then sent for incineration. Samples such as fetus are kept for burial. Empty containers are left for a week and a half as a quality control and for human errors. In many cases the label on the container shows the type of specimen so the empty containers are left in case verification of type of specimen is required. Blocks and slides are stored permanently inside a storage room. All blocks and slides are carefully and methodically filed so they are available for records or for future reference. As years pass blocks remain unchanged but stains on the slide tend to fade so there is sample deterioration. After cutting, the blocks are placed in numerical order according to the year and placed inside boxes. The first and the last number of the blocks in each box are written on the boxes. All sides and top box are labeled and sealed with tape. Slides are filed in numerical order after the report is issued. Slides are placed in a slide box and the lab number of the first and last slide are written on the box. Effective self-management of time and workload The opening hours for the histology are from 6.00 am till 5.00 pm. The lab is open from Monday till Sunday and time shifts are available so the laboratory remains more open and more service is given to the public. The laboratory does not open during night shift because results in the histology laboratory are not considered urgent. Results must first be seen by the pathologist so no immediate results are required so processing is done during the day. Samples that are considered urgent In histology, specimens are not considered urgent because they have to be viewed by the pathologist results are issued. Frozen sections are considered urgent since the sample must be quickly processed so an intra-operative decision can be taken by the surgeon. Samples can also be considered urgent when a pathologist needs the results in a quick time, due to surgery scheduled on that day or the following day. Career-Long Self Directed Learning What is CPD? CPD stands for Continuing Professional Development, an ongoing free training programme in histopathology including histology and cytology (Institute of biomedical Science, 2011). It is defined as The systematic maintenance, improvement and broadening of knowledge and skills, and the development of personal qualities, necessary for the execution of professional and technical duties throughout the practitioners working life (The Chartered Institution of Highways Transportation, 2011). This means that CPD allows the employer to improve and to widen knowledge, quality, competence and skills in his/her profession. What constitutes CPD activity? A CPD is constituted by meetings, short courses, conferences or workshops that are created to inform other members of stuff or even the public. Organization and participation are essential for a successful CPD. It must be transparent, accountable and visible (Fox Fox, 2004, p.182). CPD can be done: To present ones own research report With the aid of websites, journals, posters, books and other printed media To show something encountered during work, that can be of interest to rest of the workers To make and encourage new procedures and changes Introduce a new course that will be of interesting to the public or workers How does the CPD scheme benefit Pathology employers? A CPD scheme enables the biomedical scientist to develop the necessary knowledge, attitudes, personal effectiveness and skills for his/her professional practice. The employer must identify his/her and their employers learning needs. In order to improve patient care the employer must be up to date on facts, new concepts and most importantly on opinion and consensus (The Royal College of Pathologists, 2010). The employer can record activity and document all learning achieved (Academy of Medical Royal Colleges, 2010). All this is done not for only the present but also for future progression (Institute of biomedical Science, 2011). What are the benefits to a biomedical scientist (the employee) participating in the CPD scheme? Keep up to date with current rapid and expanding knowledge (The Royal College of Pathologists, 2010). Increases job satisfaction, productivity and quality of working life (Chen, Chang Yeh, 2006) Acquire new skills for safe and effective practice. This builds up confidence in the employee (Institute of biomedical Science, 2011). Promote professional ideas and new initiatives, increasing job satisfaction (Institute of biomedical Science, 2011). Documentation of all that is learned from the scheme is encouraged (The Royal College of Pathologists, 2010). Benefit from quality control measures (Academy of Medical Royal Colleges, 2010). Encourage reflective practice (Academy of Medical Royal Colleges, 2010). Reduce risk of clinical isolation (The Royal College of Pathologists, 2010). Prepare for new roles example managerial. Employers value employees that undergo continuous CPD since such employees show learning agility (Chen et al., 2006; Royal College of Pathologists, 2010). Maintain a reputation of the biomedical possession and public assurance (The Royal College of Pathologists, 2010). Where is the information relating to CPD displayed in Pathology? When a CPD meeting is going to be held all biomedical scientists are informed through an email. The email is sent to the principle to make sure that all the histology staff knows about the meeting. Vertical Audit Site of origin The trucut samples were taken from the right breast upper outer quadrant (Figure 2) Sample Taking and Description of sample The trucut biopsy is taken after a mammography showed a suspicious result. To diagnose, a trucut biopsy was performed. A trucut (core) biopsy is mostly done to sample tissues from a solid mass or calcium deposits, increasing sensitivity (Youk, Kim, Kim, Lee Oh, 2007). Very small masses or masses that are too deep are sampled using a guiding imaging technique. No scars are left after sampling. It has the advantage of being highly sensitive and specific (Sadler et al., 1994). The biopsy was performed at Mater Deis surgical operating theater (SOP) (in the Breast Clinic). The patient was given local anesthetic and left for a few minutes. A 16mm gauge core needle (figure 3) was then used to obtain the tissue samples. The tissues sampled contain tissues from the mass and normal healthy tissues from the breast. The sections sampled contain also provide more diagnostic information than mammography and fine needle aspiration. The samples are larger than FNA therefore results are more accurate (Kasraeian, Allison, Ahlmann, Fedenko Menendez, 2010). The clinician or nurse localised the mass and its boundaries and the mass was then immobilised. The needle was inserted through the skin into the lump and the tissue section was taken. To increase the chances of diagnosis 6 trucut specimens were taken. Their length varied from 9mm to 14 mm. The needle was then detached. The trucut specimens were then introduced into a container contain 10% buffered formalin. The container and the request form where received in the histology laboratory the following day. Specimen reception/numbering A courier brought the trucut specimen for histology processing into the histology laboratory. In the laboratory, the request form which comes together with the specimen was left for a day where registration and processing began. The following day, the receptionist used the HOE system to input data so they can be available only in the laboratory. The ID number of the patient was inputted followed by location the specimen was sampled example BOFFA, the name of the medical lab scientist, and the name of the pathologist/consultant. If available, the macro examination results were also included. A label containing the lab number, the letter on the cassette, the last two digits of the year, and the patient name and surname was prepared and printed. The label was prepared to label the slide after staining (in this case only one label was required). Specimen Registration The sample and its respective request form were both labeled with a barcode containing a specific laboratory number. The barcode was stuck on the top of the request form and at the back of the container (without covering any patients details). The laboratory number was also written with the aid of marker on top of the tap of the container. The request form was stamped at the top and at the bottom with the date in which it was received in the laboratory. The trucut specimen and the other histological specimens were left one after the other, according to the laboratory number. Specimen processing proceeded in this order. Specimen Processing 1. Cut-Up The trucut specimen was first processed in the laboratory at the cut-up laboratory. The name and surname of the patient and the lab number on the request form and on the specimen container were checked. The trucut biopsies in 10% buffered formalin were taken out from the container, using forceps, on the working bench. A macroscopic examination was performed on the 6 trucut biopsies obtained. Their length ranged in length from 9mm to 14mm. They were all embedded in one printed cassette labeled A1. Blue foam was also placed and the cassette was covered with a medal lid. It was then was placed in eosin with the other specimens. The trucut biopsies were then ready for further laboratory processing. After all specimens were cut, a histopathology worksheet was filled in. This included the case number of the patient, the number of tissues taken (6) , the tissue type (breast trucut), the number of blocks (A1), any comments such as left to fix (not applicable), the name of the pathologist who will examine the slides, and the name of the medical laboratory scientist (in this case who performed the cut up). 2. Tissue Processing (Impregnation) The biopsies were processed in an automated processing machine. This was performed in a closed system for trucut specimens using program A. It is important that the specimen is not larger than 3mm since it will not fit and cannot be cut afterwards. The closed system has 14 baths and it provides pressure, waving, bubbling and rotation to the tissues so the reagent can penetrate better. This is performed overnight, therefore the processor is programmed. When time for embedding is prolonged, the fixation time is prolonged to compensate. The tissues were first fixed in 10% buffered formalin so that the fixation was continued They were then dehydrated in 2 baths of 70% alcohol, in 1 bath 95% alcohol and then in 2 baths of absolute alcohol. The dehydrated sections were then moved into the chloroform and xylene. This step was done for clearing. Chloroform is a carcinogen and it affects the nervous system. The tissues were automatically moved in wax for tissue impregnation. This caused the tissues to harden. A temperature of less than 60oC was necessary because a higher temperature would have affected elasticity of the wax. Fumes go in a waste bottle and charcoal filter is present to filter leak. The tissues were now ready for embedding. 3. Embedding During embedding, the formalin fixed processed tissues are surrounded by wax so a solid paraffin block is obtained. This will enable the medical lab scientist to obtain thin sections from the block so that they can be stained and later viewed by the pathologist. The procedure involved was as follows: The cassette was taken from the processor to the warm compartment of the histocentre. The histocentre is an embedding center that facilitates paraffin embedding. It is equipped with a dispenser, specimen handling tank, warmed embedding moulds, warmed forceps wells and warm plate for orientation of the specimen in the melted paraffin. After checking the wax tank was properly filled, the cold plate and light were switched on. The cold plate helps in transferring of the melted paraffin. The tissue cassette was opened and the number on the labeled cassettes was checked with that on the worksheet entry. A suitable mould compartment corresponding to the size of the tissues in the cassette was chosen. The mould was filled with paraffin wax The tissue was placed at the bottom of the mould, correctly orientated. Incorrect orientation ruins the first section taken The trucuts were placed centrally aligned across long axis of the mould, and not placed at random. Adequate border of embedding medium must surround all sides of the tissue to give maximum cutting support. The mould placed in its cassette was placed on a cold plate and allowed to solidify. The block was scraped along a para trimmer to trim excess wax on surface. Tissues embedded must be perfectly flat to ensure that a complete section will be obtained. 4. Microtomy After the block was trimmed, thin sections were now cut using a microtome. The block was first trimmed to expose the area to be sectioned. A sharp non-rusted blade was used not to cause damage to the tissue by scoring. The microtome was cleaned from staples and sutures that remained to avoid damage of the blade. Microtomy was then started. The tightly screwed blade was checked and adjusted in the correct position. The micrometer gauge was set at a thickness of 18-22m. The block was placed inside the block holder of the microtome and secured. The block holder was in parallel to the edge of the blade so a straight ribbon of sections was obtained. The block holder was moved using the couae trimming device until the wax block was almost touching the edge of the blade. The fine trimming rotator device was when the block touched the edge of the blade and trimming of the block was started. Excess wax from the surface of the block was removed until the surface of the tissue was exposed. Debris due to coarse cutting was removed using a Camel hairbrush. The block was then placed on ice to cool giving the tissue and the wax similar consistency. Water absorbed by the tissue, slightly swelling it, so cutting is easier later on. If this does not occur sections tend to crease. The block was reattached to the microtome, leaving the left-hand rotator device. The micrometer gauge was then set at 3m. A series of sections forming a ribbon were cut and the first one was not used since it is usually thicker than 3m. 4 layers were taken. This means that the after the first section was achieved, the next few layers were ignored and then a second section was taken. The same was done for the third and fourth. This is done so that the pathologist can study many layers from the site taken so that diagnosis is more accurate. The appropriate ribbon section (for all the four sections obtained) was gently transferred into a water bath using forceps. The water bath is set a few degrees below the melting point of the wax. The sections were floated onto a glass slide containing 20% alcohol. The ribbon section was then released on the surface of a water bath (at a temperature less than that of melting point of wax i.e. 60oC. The sections were collected on an APES-coated glass slide. They were placed on near the other. Coated APES facilitates adhesion of the sections onto the glass slide. 4 slides were obtained (a slide for each layer taken). The number of the block was written on the glass slide using a diamond pen and placed on a slide rack. It was dried in an oven at about 60oC for 10 minute. 5. Staining Together with the other blocks from the other specimens the slides were dried and now ready for staining. The routine gold standard stain in histology is Haematoxylin and Eosin stain. This was done in an automated staining machine which followed the regressive method. This allowed overstaining of the tissues and removal excess dye by differentiation. The staining procedure was programmed as followed: The slides were left in the heating station so that all water is removed. The slides were dewaxed in a xylene for 4 minutes. This removed the surrounding wax from the tissues. They were then placed in xylene alcohol for 15 seconds. This started the gradual hydration process and prepared the tissues to be stained by haematoxylin dissolved in an aqueous solvent. The hydration process is followed by 2 baths of absolute alcohol (15 seconds each). The slides were then passed into four baths: 95% alcohol, 70% alcohol, 50% alcohol, and 30% alcohol (15 seconds each). They were then passed for 15 seconds in distilled water since haematoxylin stain is water based. The slides were then passed for 10 minutes in haematoxylin stain. The time inside the haematoxylin bath varies according to the properties of the stain. The prolonged use of the stain increases the time the slides pass in the bath. The slides were rinsed in two baths of distilled water, the first bath for 30 seconds and the second bath for 10 seconds. Differentiation then occurred in acid alcohol for 1 second. This allowed the nucleus to retain the stain and to decrease the pH (acidic) so colour changes to light purple. The slides were rinsed in distilled water for 15 seconds. Bluing occurred in tap water for 5 minutes. This raised the pH so sections became light blue. The slides were then passed into a bath containing distilled water for 15 seconds. The slides were passed in absolute alcohol for 15 minutes for dehydration and because this favours alcoholic eosin staining since it is alcohol based. Counterstaining was performed in a bath containing alcoholic eosin for 3.15 minutes. The time in alcoholic eosin varies according to the properties of the stain. The prolonged use of the stain increases the time the slides pass in the bath. Prolonging time allows the cytoplasm to take up the pink eosin stain. The slides were then dehydrated in four baths of acid alcohol, 15 seconds each. The slides were cleared in xylene alcohol for 15 seconds followed in 2 baths of xylene for 5 minutes each. This helps during mounting since DPX mountant is xylene based. The slides were left in the heating station so that all water is removed. The slides were taken out from the rack and mounted with DPX mountant. Quality Control: Two slides are stained with H E stain using the automated machine in the morning before starting routine staining. Errors in staining such as weak stains and contamination (example of eosin) can be detected so they can be solved. The 4 slides of the patient were therefore well stained since the machine passed QC on that day. Results: Nucleus: Blue Cytoplasm and other eosinophilic structures: Pale pink After processing, the number on the slides was checked with that of the cassette and the block. The slides were then labelled with their respective label. The cassette was placed on top of the slide to see if all the stained sections present on the block were sectioned. All the stained sections agreed with those on the block. Role of the Biomedical Scientist The role of the biomedical scientist is to perform all the above procedures. The medical lab scientists are divided into different sections throughout the histology laboratory: in the cut-up room and in the embedding and staining section of the laboratory (excluding immunohistochemistry laboratory). In addition, the biomedical scientist must also fill several worksheets. The initials of the biomedical lab scientists performing the cutup, macro-examination, LID and embedding are written in the histopathology worksheet. The MLS must monitor any changes example in reagents. Any injuries or misshapen occurring in the laboratory must be recorded. Pathologist Role/Result Reporting After staining, the pathologist viewed the slides under the microscope and performed a microscopic examination. The observed results were noted. The microscopic examination results were sent to the secretary who typed the result in the results form. The pathologist then read the results form for any errors and once the result was verified the pathologist authorised the result. Result Entering and Authorisation After the pathologist viewed the slides under the microscope he took the fully written request form to the secretary. The secretary separated the forms into different piles, according to the pathologist. The form was typed in a result form and printed as a result sheet. The written and the print result form were separated into 2 different racks. The report sheet was taken to the respective consultant/pathologist who reviewed the printed result sheet for any mistakes. This includes patient details, clinical details, and examination results. Once the pathologist verified the data written, he used the software to authorise the result. Once the pathologist authorised the result, this was available in the LIS of the cytology and histology laboratories. The CMI system allowed the results to be available to the wards. The result sheet was taken to the secretary where the result form was piled with other results forms according to the pathologist/consultant. Copies were made and sent to ward and patient. Result Issuing (Describe the results form) The results form contains the details of the patient, including the name and surname, address, date of birth, sex and the hospital number. The name of the clinician and the site from where trucut biopsy was taken (SOP) are included. The date the specimen was taken and the date and time it was received are also included. The lab number associated to the specimen is important to be included because besides identifying the patient it can be used for future reference. If the slides or block containing the sections are required they are labelled (including lab number) and stored and easily retrievable. The specimen type and site from where the biopsy was taken, the macroscopic examination and the microscopic examination are all included. The included, in this case Benign breast parenchyma of the right breast. The pathologist and the date and time the result was reported and authorised (by pathologist) and the date and time the result form was printed are also included. Benign Breast Parenchyma: The breast parenchyma forms part of the normal breast tissue. It was reported as benign during microscopy because of few scattered (not clustered) lobules seen in breast sections. Since no atypical features were observed, no special stains or immunohistochemistry staining (example ER or Her-2 stains) were required. It is ideal the patient undergoes regular breast screening. Sample Collection and Specialist Preparation The containers to process routine surgical specimens vary from small to large received in 10% buffered formalin. Very large containers are rare. The container used depends on the size of the specimens. Small specimens such as polyps, prostate scrapings, appendix, trucuts, and trephines are received in small containers containing 10% buffered formalin. Some specimens such as fetus vary in size such as fetus and colon so they received in larger specimens (medium when compared to small containers). Large specimens such as lung, breast, and colon are received in large containers containing 10% buffered formalin. Large specimens require more than one day to be cut. First the specimen is opened and left for an additional day or more for further fixation. The following are types of specimen the laboratory receives that require specialist preparation techniques and the actions taken: Trephine and Bone specimens: Decalcification with EDTA or formic acid. EDTA is used example for bone marrow trephine and formic acid is used example on bone sternum for one day Figure 4 showing a femur bone undergoing decalcification in EDTA. Infective specimen example with HIV Over fixation in formalin to kill infective cells* Lymph node The time of fixation depends on the thickness of the specimen. More time the more the fixative is allowed to penetrate the lymph node.* It is left for two or three days depending on the thickness of the specimen. Over fixation will destroy the surface antigens causing artifacts and a false negative result during immunohistochemistry. Sural nerve: Sent from operation inside a gauze soaked with saline. The request from and case summary are required. The cut up laboratory gives the lab number and send the specimen to the immunohistochemistry laboratory. The tubular sural nerve is wet, and the two ends of the nerve are cut. One end is sent to a pathologist to get an idea of diagnosis and the centre part of the nerve and the other cut end are sent abroad. Muscle: This is received in saline and a lab number is given in the cut up laboratory and then sent to immunohistochemistry laboratory. It is frozen at -70oC and cut by a cryostat at -20oC. The thin sections are then stained with a series of special stains example Oil Red O and with immunohistochemistry stains example myosin. APES coated glass slides are used to prevent the tissue section from sliding off. Imprints: Example lymph node: A slide is pressed on the lymph node and the imprint is sent abroad. The lymph node is then worked normally in formalin. Imprints are used for genetic studies. Liver with no tumour: A series of special stains are performed: PAS useful if there is a high glycogen content upon staining Reticulin Stain useful in liver cirrhosis and liver fibrosis Massons Trichome Stain Useful in liver fibrosis Iron Stain useful for haemosiderosis, haemochromatosis Title: Frozen Sections Aim Performing a macroscopic examination by the pathologist Cut up of the specimen Obtaining sections at -17oC using a micrometer, inside a cryostat Staining the section/s by haematoxylin and eosin stain Performing microscopic examination of the stained section/s by the pathologist Introduction A frozen section is a specific type of biopsy performed during surgery so that a rapid diagnosis of the tissue extracted is made (Brender, Burke Glass, 2011). The tissue can be sectioned and stained in the laboratory for microscopic examination by the pathologist. The surgeon is given flexible intra-operative decision making according to the result given by the pathologist after the rapid processing (Karciolu, 2005, p.121). Principle A surgery is booked and a biopsy is taken and sent to the laboratory. As soon as the fresh specimen arrives in the histology laboratory the pathologist and the selected biomedical scientists start processing the specimen. The pathologist performs a macroscopic examination on the specimen and the observed features are written down by the pathologist. The MLS then start cutting thin sections according to the specimen, using a microtome inside a cryostat at -17oC. The sections are then quickly stained with haematoxylin and eosin stain. In contrary to routine H E, the sections are not passed through xylene and dehydrated down to water. This is because the frozen sections are not embedded in paraffin wax prior staining. Since the stain is very fast there differentiation with acid alcohol is also not performed. After mounting the pathologist checks if the stained slide is satisfactory and after performs a microscopic examination. This lets the surgeon decide what to do next. Materials and Equipment required Cryostat, OCT medium, cryospray, Glass slides, cover slips, disposable pipettes, Procedure 1. Macro-examination The pathologist opens the container/s containing the specimen/s. A macro examination is performed on the specimen/s and the pathologist starts a description so that the medical lab scientist writes on the request form. The description includes the size dimension (length x width x height) in centimeters, the shape of the specimen and if it is soft or hard. The consultant suspects carcinoma and sampling is them performed. 2. Cutting the specimen The consultant cuts piece of the specimen that covers the whole area of the specimen. It is important the most suspicious is included in the segmented section so that the consultant can find and detect the tumour during microscopy. If required, multiple sections can be taken to make a diagnosis. The size cut depends on the size of the sample and tumour. More than one pieces of the specimen can be cut example: two sections from a liver (due to liver transplantation), and from a lymph node attached to the liver. 3. Cryostat The cut specimen/s is/are placed, with the aid of tweezers, in the center of a cryostat object disk containing OCT medium. The cryostat object disk with the tissue is placed on the cryobar (holder) inside the -17oC set cryostat. The tissue is left to settle so it gets cold and this is enhanced by using a cryospray. When the tissue solidifies it is placed onto an object disk holder. The machine is set at 5 on the control panel and the block is moved towards the edge of the blade. After making sure it is properly clamped trimming is started. The rotator on the right of the cryostat is turned. The section begins to curl as the block comes in contact with the blade. The section is held down slowly and gently with tweezers and cut until the surface of the tissue is visible. The cryostat is now quickly set at 30 (this is the thickness used for most of the specimens in histology). A good section is detached and taken onto a glass slide placed opposite of the block. As the tissue comes in contact with the glass slide it sticks onto it since it melts and adheres to it. The glass slide is immediately in the staining station found adjacent to the cryostat. Haematoxylin and eosin staining is performed. 4. Haematoxylin and Eosin Staining The glass slide with tissue section is fixed in formalin for 10 seconds The slide is then rinsed in water (10 dips) It is then stained in haematoxylin (15 dips) The section stained with haematoxylin is rinsed in water Bluing is then performed by dipping the slide 15 times in Scots The slide is rinsed in 95% ethanol (10 dips) The section is counterstained in alcoholic eosin (10 dips) The stained section is dehydrated in absolute alcohol The slide is cleared in 3 baths of xylene The slide is immediately mounted with DPX 5. Microscopic examination: The stained section is immediately taken to the consultant who verifies if the section taken is appropriate or not. If the section is not appropriate a new section must be cut and stained (a second section is cut to play safe because time is precious for the patient and for the surgeon to make a decision). The consultant sees the stained tissue section under the microscope and sees the previously suspected tumour. If more than one slide are required due to a lymph node the consultant checks if there are tumour cells due to metastasis. The surgeon is immediately informed to see what to do next. Quality Control The slides are seen by the pathologist as quickly as possible. If the slides are not good then the pathologist orders other sections, example order thinner sections of sections with less stain intensity. More than one section is stained during processing of frozen sections so that if the pathologist orders a new slide, it is already available. Precautions Microtome should be working with a clean sharp blade knife Blade angle should be between 30 to 50 The anti-roll plate should be adjusted to prevent curling of the section. This means that blade edge must have correct height and angle, plate edge should not be damaged and the cabinet temperature must be correct sections. A sable hair brush manipulates section when anti-roll plate is not functioning properly (Bancroft Gamble, 2008, p.100) The blade should have sharp edges to obtain good quality sections (Bancroft et al., 2008, p.100) Optimum temperature according to the type of specimen being cut. Most tissues are cut a temperature maintained at -20oC 5C. Liver, brain, lymph node, spleen and uterine curettings which are sectioned at -10C. Fat and breast with fat are cut at lower temperatures (-25o to 30oC). Tissue must be fresh not dehydrated Cryospraying is not done directly on the section (especially if it is a thyroid section). This causes artifacts (round circles) When there are cutting problems the microtome is defrosted and maintenance is called (Bancroft Gamble, 2008, p.100) Results: Nucleus: Blue Cytoplasm and other eosinophilic structures: Pale pink Advantages of frozen sections Additional samples can be taken for additional samples. This avoids a second surgery (Brender et al., 2011) Quick diagnosis can be made (Brender et al., 2011) Cancerous tissue can be removed at the time of injury. Benign mass do not always need to be removed (Brender et al., 2011) Ensures that the removal of the tissue is that of the intended tissue (Brender et al., 2011) The whole mass and its boundaries are removed (Brender et al., 2011) Scientific research can be performed on the tissue samples (Brender et al., 2011) There is cooperation between patient and pathologist so that the patient can benefit as much as possible (Brender et al., 2011) Multiple regions from a single set of sections can be quantified (McKim et al., 2004) Costs are reduced (McKim et al., 2004) Time is reduced when few sections are required and when sectioning and staining go smooth (McKim et al., 2004) Limitations The time for the result can increase as the number of frozen sections required increase. The patient remains more under surgery. Complex and large specimens take more time. Therefore, sectioning and staining is rapid for intra-operative decision by the surgeon (Peters, 2009, p.14). When compared to permanent paraffin sections, frozen sections contain more artifacts. Sectioning and staining defects including wrinkles, non-smooth incomplete sections (lack of margin control), folds, staining condensations and cellular distortion (Karciolu, 2005, p.121). Poor sampling can cause inappropriate diagnosis (Karciolu, 2005, p.121). Very hard samples (calcified) cannot be cut and sectioned therefore they cannot be rapidly processed. A lower temperature in the cryostat produces a harder block and a higher temperature produces a softer tissue. Lack of consultation between biomedical scientists during processing Diagnostic Application Intraoperative diagnosis The sections are rapidly obtained so surgeon can decide what to do. Other applications include: Non-enzyme histochemistry demonstrates unsaturated lipids example in analysis of lipids of arterial walls, fetal colon, lung and muscle (Liadsky Woolf, 1967; Garbarsch, 1969). Protein-bound lipids removed from the tissue during routine processing of paraffin sections (Bancroft et al., 2008, p.191). Frozen sections can be stained for lipid by Oil Red O (Tracey, Kissling, Gandia Reynolds, 1989). Non-enzyme histochemistry: Frozen section can also be used to demonstrate mucopolysaccharidoses example in bone. These carbohydrate deposits are weak to formalin so there is little preservation of sections (Bancroft Cook, 1994, p.146). They are water soluble so special fixatives or frozen sections are performed (Stocker Dehner, 2001, p.186). Enzyme histochemistry: Muscle biopsies require enzyme methods to produce a definite diagnosis. A temperature of -70oC allows little enzyme loss so the muscle fibers are more preserved than they would be in fixation. Enzyme histochemistry allows a more and detailed localised reaction of the histochemical reaction product (Murray Ewen, 1989). Immunohistochemistry (IHC) and Immunofluorescence (IMF): Antigens are inactivated during fixation but preserved during preparation of frozen sections for IHC and IMF. Infective agents are also destroyed during preservation of the substrate antigens (Bancroft et al., 2008, p.151). Examples of tissue preparation for antigen demonstration are classification of lymphoproliferative disorders (IHC) and skin and renal biopsies (IMF) (Sheibani Winberg, 1987; Bancroft et al., 2008, p.518). Neuropathology: Can be used on central nervous system sections which are then stained with silver stain (Bancroft et al., 2008, p.98). The sections are sent wrapped inside a gauze in saline during surgery and cannot be fixed. Preservation of DNA and mRNA for molecular biology. Usually when fixed with formalin protein links between mRNA and proteins occur if left for more than one day. Alternative Methodologies Slow mohs: It is a method used for skin cancers such as lentigo maligna. After excision is performed the patient is sent home. A permanent paraffin section is made from full cuts of the specimen. In the paraffin block, the epidermal and the deep margins are orientated in the same plane (Rutledge Chlipala, 2004). This helps to spare normal tissue. The patient comes back for a second excision only from those areas that are positive. This goes on until microscopic examination shows tumour clearance (Huang, 2004). Frozen sections are quicker and a decision can be done within minutes so, example, a tumour can be completely removed. Freeze Drying: Sections are quenched and dried. But these sections are allowed to reach temperature before fixation. Therefore, they take more time than frozen sections (Bancroft et al., 2008, p.102). Conclusion Frozen sections allow rapid processing and rapid microscopic examination of a biopsy/ies taken during surgery. Although it has a lot of limitations, it allows intra-operative decision making to the surgeon. Title: Immunohistochemistry Aim Cutting tonsil sections using a microtome for immunohistochemistry Performing antigen retrieval using sodium citrate buffer (pH 6.0) Performing immunohistochemistry on the sections, using the Avidin-Biotin Complex method Introduction Immunohistochemistry (IHC) is an advanced technique used to detect antigens on cellular or tissue constituents by means of antigen-antibody interactions. The site the antibody (marker) binds can be identified by directly labeling the antibody or by using a labeled secondary antibody (Bancroft Gamble, 2008, p. 435). Visualization can be made under light microscopy. IHC is important in diagnosis when a therapeutic decision for neoplasia leads to inconclusive results (Hayat, 2006, p.532). Principle After the sections are cut, using a microtome, the slides are collected on labeled APES coated glass slides. These glass slides enhance adhesion of the section on their surface and prevent the section from getting lost due to high temperatures. The slides must be dried before they are introduced in xylene (to prevent contamination). Antigen retrieval Fixation with neutral buffered formalin causes the formation of methylene bridges that cross-link proteins and cause masking of the antigenic sites. To retrieve antigens, a heat-induced epitope retrieval method with sodium citrate buffer (pH6.0) was performed. The buffer is placed inside a pressure cooker, followed by the slides (inside a metal rack) after the buffer boils. The methylene bridges break and the antigenic sites are exposed allowing binding of antibodies. The addition of diluted hydrogen peroxide to the slides will block enzyme activities of the tissue. Cells such as red blood cells contain endogenous peroxide that is able to react with the developing mixture. By pretreating tissues with hydrogen peroxide will prevent non-specific background staining when adding the enzyme (HRP) (Li, Ziesmer Lazcano-Villareal, 1987). Slides are adhered onto a cover slide and mounted inside a mounting chamber. Normal swine serum is added to block non-specific binding of antibody to the antigen on the tissue. The primary antibody (polyclonal anti-CD3) containing rabbit polyclonal then was left overnight, to bind with the CD3 antigen on the surface of the tissue section. Excess antibody is removed by washing with PBS without suppressing the antigen-antibody interaction. The solution also prevents non-specific binding and maintains a pH 7 environment. The addition of Biotinylated Goat Anti Rabbit (secondary antibody) is directed against the primary antibody. It is left to bind for 1 hour and excess unbound secondary antibody is washed, using PBS. ABC (Avidin-biotin peroxidase complex) is able to create a 3-D that contain a lot of biotinylated horseradish peroxidase molecules (HRP enzyme) that are cross-linked by avidin. Enzyme is located at the antigen site through these complexes, increasing sensitivity (Bratthauer, 1999). Glycoprotein Avidin has four binding sites per molecule (i.e. a high affinity for biotin) and biotin is able to bind with only one binding site with avidin. It is able to bind irreversibly to the secondary antibody. To the DAB solution, hydrogen peroxide is added before it is added on to the sections. DAB/H202 acts a colorimetric substrate solution. HRP catalyzes the hydrogen peroxide and electron transfer from DAB to HRP occurs to yield a brown insoluble product. The color intensity is seen under low power and stopped by phosphate buffered solution. When there is background the reaction is stopped immediately. Counterstaining is performed with haematoxylin since CD3 is a cytoplasmic and a membrane stain. This means that the counterstain will stain the nuclei blue. The sections are dehydrated, cleared and mounted and viewed under the microscope. Materials Required Glassware rinsed in distilled water, single channel pipettes, cover slips, disposable pipettes, slide rack, Pressure Cooker, pH meter, Microtome, Oven set at 60C, Microscope. Reagents and Preparations Citrate buffer was prepared by adding 3.15g of citric acid and 1500mls distilled water Hydrogen peroxide 3mls 30% H2O2 and 300mls of distilled water Normal swine serum (NSS) A dilution of 1/20 was prepared. This required adding 250l of CD3 antibody and 4750 l of antibody diluent to make a total of 5000 l. Primary antibody (rabbit polyclonal to CD3) This required a dilution of 1/350. Since 5 slides were required, (one slide for this case and 3 routine slides and a control) 2 l of antibody and 698 l of antibody diluent were added. Biotinylated Goat Anti Rabbit (BGAR) A dilution of 1/300 was prepared. This required adding 16 l of concentrated BGAR and 4784 l of antibody diluent to make a total of 5000 l. Antibody diluent is made of made of albumin, sodium azide and phosphate buffered saline. Dilute Phosphate buffered Saline was prepared by adding 200mls of concentrated phosphate buffered saline and 1800mls of distilled water. Avidin-biotin peroxidase complex (ABC) solution: This was prepared by adding 50 l of avidin and 50 l of biotin to 2400 l of phosphate buffered saline. 3,3-Diaminobenzidine (DAB) A solution was prepared by adding 1 tablet of 3,3-Diaminobenzidine tetrahydrochloride stored in freezer 1 tablet of Tris-buffered saline 15mls of deionised water After left to dissolve, it was filtered and later 12l of hydrogen peroxide (H2O2) were pipetted (freshly prepared before use) Commercially prepared Haematoxylin, xylene, xylene alcohol, absolute alcohol, 75% alcohol, 50% alcohol, DPX mounting Medium Procedure (including precautions) Day 1 After the request form for immunohistochemistry was registered and the respective block was retrieved, the block was placed with the section touching the ice. One slide was marked with the patient number, date and test required, in this case CD3. Another slide was used as a control. (In total there were 5 slides for CD3 since one slide was used for this case, another was the control, and the other three were other slides from 3 different patients) The block was placed in the block holder of the microtome and the appropriate angle was found (so that there is one whole cut). The ribbon of wax containing the sections was then placed inside a water bath at 37oC and a section was collected on every slide. The slides were dried inside an oven at 60oC. Water would have contaminated the xylene during deparaffinisation. The measuring probe of the pH reader was inserted inside a liquid of pH 7 that read pH7.08. This served as a control, therefore it was working properly. The citrate solution was prepared and sodium hydroxide pellets were added. The measuring probe was immersed and the rise in pH was noted: pH6.10 The citrate buffer solution was then placed inside a pressure cooker. At the same time the dried slides were depraffinised in xylene for 2 minutes. The slides were then left for 2 minutes in 2 other xylene baths. The slides were then placed in xylene alcohol for 2 minutes, to start the hydration process. Hydration was then continued in a bath containing 75% alcohol, and a bath containing 30% alcohol, each for 2 minutes. The slides were then immersed in water. The pressure cooker, at the mean time, was powered on mark 4 until there was full pressure. It was left 3 minutes at full pressure and then adjusted left on mark 1. The slides were incubated with the buffer, inside the pressure cooker for 10 minutes for antigen retrieval. The pressure cooker was then placed inside a sink, the pressure release valve was activated and cold water was run outside the lid and then inside, for 10 minutes. The slides were treated with 12l hydrogen peroxide for 10 minutes, to block enzyme activity of the tissue, and washed. The slides were than attached to chamber slides under water (with the side of the section touching the chamber slide). They were tightly held and placed inside a mounting chamber. The slides together with chamber slides were placed in a straight position so that fluid reached the whole section and not part of it. This would have stained only one part of the section. The control was first placed and followed by the slides to be tested (CD3). The slides were washed with PBS. If it drained quickly, the slides and the cover slides were not attached appropriately. 100 l of NSS were pipetted between the chamber slide and the slide with the section (tightly adhered together). It was left for 12 minutes so the next reagent does not dilute. Without rinsing, 100 l of the primary antibody were pipetted and left overnight refrigerated at 4oC. Day 2 The mounting chambers were removed from the refrigerator and all slides adhered to chamber slide were washed with PBS. This covered the whole section. The PBS was allowed to drain. 100 l of BGAR were then pipetted and left incubated for one hour on the working bench, closed with the mounting chamber lid Washing of sections was then performed using PBS. 100 l of ABC reagent were pipetted and incubated for 1 hour on the working bench, closed with the mounting chamber lid The sections were washed with PBS The slides were removed from the cover slides under water to prevent damage of the section. The slides were placed back to back on slide racks so that one section does not touch the other. They were placed in a bath containing PBS. 12 l of H202 were added to the DAB solution. The back and the sides of the slides were dried with a clean tissue and the section was covered with DAB/ H202 working solution. The slides were immediately observed under the microscope at lower power to assess the development of the colour. If the brown colour in the control is satisfactory it is stopped by dipping the slide in PBS. The rest of the slides for that same test are also stopped in PBS. If there is slight background in the section the slide is immediately stopped in PBS. The slides were rinsed in water. The slides were then counterstained in haematoxylin for 1 minute and placed in water bath. They were then placed in warm water. The slides were dipped 6-7 times in alcohol and rinsed with water. Bluing was performed in tap water for a few seconds The sections were viewed under the microscope to check that the nuclei were blue Dehydration was then started by placing the slides (2 minutes each) in 30% alcohol, 75% alcohol, and in 2 baths of absolute alcohol for 2 minutes each. The slides were cleared in xylene alcohol for 15 seconds followed in 2 baths of xylene (2 minutes each). This helps during mounting since DPX mountant is xylene based. The clearing agent is necessary because dehydrating agents are not miscible with impregnation medium. It acts an intermediate chemical. This removes alcohol. The high refractive index makes the tissue clear so that this is equal to that of the DPX mounting medium. The slides were mounted in DPX mounting medium and allowed to dry. Quality Control In immunohistochemistry a different control is used for every different stain test required. In this case, a tonsil section was used as a positive control for all CD3 tests required. Results: Cytoplasm and membrane Brown Nuclei Blue Interpretation of Results Control (Tonsil): The cytoplasm and the membrane stained brown, the nuclei stained blue. Therefore the control worked and showed diffuse positivity. Section (Patient tonsil tissue section): The cytoplasm and the membrane stained brown and the nuclei stained blue. But staining was not diffuse because there were areas in the cytoplasm and membrane that did not stain. The problem encountered could be that the DAB was not given a lot of chance to stain so the stain was very weak and areas did not stain. The stain was not very crispy, this is mostly possible because the section was big and not of very good quality. There was little staining in the mantle zone, and weak or very weak staining in the germinal center. The paracortical and interfollicular areas stained well. The capsule did not stain as expected. The CD3 antigen CD3 antigen is part of a polypeptide chain complex found on the surface membrane of T-lymphocytes, associated with the T-cell receptor (TCR). CD3 , CD3, CD3 and CD3 are all CD3 molecules forming part of the TCR and are stained with CD3. It is involved in signal transduction (Ioachim Medeiros, 2008, p.53). CD3 stains the cytoplasm and/or cell membrane (Law et al., 2002). Cytoplasmic positivity is in the early and late stage of development of thymocytes and membrane positivity is shown in T-cell lymphomas (Wang et al., 2009). CD3 stain can stain T-lymphocytes mostly in peri-follicular areas and to a lesser extent in germinal centers, mantle zones, stratified squamous epithelium and loose connective tissue (Harris, Meghji Speight, 1997; Ioachim et al., 2008). Diagnostic Application Besides normal tissue, CD3 stain is used to identify T cell neoplasms example: T-Cell Lymphomas and/or Natural Killer Cell lymphomas: T cells express CD3 so membranous staining is specific for T-cell lymphoma. Natural killer cells are able to show epsilon expression on their cytoplasm (Chu Weiss, 2009, p.486). Mycosis fungoides: A major subtype of cutaneous T-cell lymphoma (Pimpinelli et al., 2005). T cells are dominant in the dermis and epidermis. Infiltration of T-helper memory cells are a characteristic of mycosis fungoides (Cerroni, Gatter, Kerl Helmut, 2009, p.24). Alternative Methodologies Catalysed signal amplification Used to visualise rare or masked antigens that show weak Immunohistochemical signals. It is time consuming, and staining is complex and poorly reproducible (Hashizume, Hatanaka, Kamihara Tani, 2001). Peroxidase anti-peroxidase method (PAP) This is a 3 layer method considered sensitive (not as much as the ABC method) and eliminates non-specific binding. On the other hand, it has some disadvantages: primary antibody and PAP are raised from the same species, and it is expensive to obtain readymade complexes from different species (Bratthauer, 1995). ImmPress method: After addition of the primary antibody, there is addition of ImmPress reagent followed by peroxidase substrate. The result is fast due to the reduced incubation steps. It is very sensitive and produces discrete localisation of the antigen. Background staining is reduced because it pre-diluted (Vector Laboratories, 2010). Conclusion Immunohistochemistry is a very important technique in histology that demonstrates the localisation of the antigen on the surface of tissues. It has the advantages of efficiency (not time consuming), high sensitivity, stability and versatility and low background staining due to high pre-dilution. Although a positive result is obtained when an antibody is targeted towards the antigen, IHC is unable to define distinct cell populations. This can be achieved by flow cytometry. List of Hazardous Reagents Equipment Name: Year of Entry: Task: Date: Substance Associated Risk Actual risk to user Safety measures Action taken in case of incident Methylated Spirit Flammable Toxic Fire Eye Splash Ingestion Store in flammable cupboard Use PPE Eyes wash with water for 10min Skin wash with water Ingestion wash mouth with water, seek medical attention Chloroform Toxic Eye irritation Skin Irritation Ingestion Inhalation Store in safety storage cabinet away from heat and sources of ignition Eye wash station in the laboratory Use PPE Seek medical attention Eyes wash with water for 15 minutes Skin Wash with plenty of water and soap. Cover irritated skin with an emollient Ingestion Do not induce vomit, loosen tight clothing Inhalation rest in a well ventilated area 3,3-Diaminobenzidine tetrahydrochloride (DAB) Tablet (DAB) Tablet (continued) Flammable at high temperature Toxic Fire (at high temperature) Eye irritation Skin Irritation Ingestion Lung Irritant Store at -20oC in the dark Use PPE Seek medical attention Eyes wash with plenty of water for 15 minutes Skin Wash with plenty of water and cover irritated skin with an emollient Ingestion Wash out mouth with water Inhalation rest in a well ventilated area DPX Mountant Flammable Toxic Fire Eye Splash Skin Irritation Ingestion Inhalation Store in tightly closed labeled containers, store in a well ventilated area away from ignition Use PPE Seek medical attention Eyes wash with water for 15min Skin wash with water Ingestion wash mouth with water Inhalation move to fresh air away from exposure Ethanol Flammable Toxic Fire Eye Irritant Skin irritant Ingestion Inhalation Store in a segregated and approved area. Keep in a non-ventilated area tightly closed and not above 23oC Use PPE Seek Medical Attention Eyes flush with water for 15 min Skin Wash with plenty of water and cover irritated skin with an emollient Ingestion Do not induce vomit, loosen tight clothing Inhalation move to fresh air away from exposure Eosin Yellow Eosin Yellow (continued) Toxic Carcinogen Eye Irritant Mild skin irritant Digestive Tract irritation Inhalation (Poisonous) Store in a cool, dry, well-ventilated area away from incompatible substances. Use PPE Seek Medical Attention Eyes flush with water for 15 min Skin Wash with plenty of water and soap. Remove contaminated clothing. Ingestion Do not induce vomit, loosen tight clothing and give water Inhalation move to fresh air away from exposure Formalin buffered Solution Toxic Flammable when liquid vaporizes in air Corrosive Fire Eye Splash Skin Irritation Ingestion Inhalation Safety Storage cabinet room store in a well ventilated area away from ignition Use PPE Seek medical attention Eyes wash with water for 15min, Skin wash with water Ingestion wash mouth with water, seek medical attention Inhalation move to fresh air away from exposure Freezer Spray Flammable (only when heated) Toxic Explosive (only when heated) Eye Splash Skin irritation Ingestion Inhalation Store in a well cool ventilated area away from light Use PPE Seek medical attention Eye wash with water for 15min Skin Wash with cold water and soap, remove contaminated clothing Ingestion Do not induce vomit, loosen tight clothing Inhalation: move to fresh air away from exposure Harris Haematoxylin Toxic Eye Irritation Skin irritant when in long contact GIT irritant Inhalation Store in a cool place and out of direct sunlight and heat. Use PPE Seek medical attention Eye wash with water for 15min Skin Wash with cold water and soap, remove contaminated clothing Ingestion Do not induce vomit, loosen tight clothing Inhalation: move to fresh air away from exposure Hydrochloric acid (HCl) Flammable (negligible) Toxic Corrosive Fire (negligible) Eye splash Skin irritation Inhalation Store in a dry cool area Use PPE Seek medical attention Eye wash with water for 15min Skin Wash contact areas with soap and water, wash contaminated clothing Inhalation: move to fresh air away from exposure Paraffin wax/Paraffin wax fumes Combustible at high temperatures Toxic (Acute) Fire at high Temperatures Eye irritant Skin irritant Inhalation Ingestion Store away from heat and ignition. Store in a dry cool place. Seek medical attention Eyes flush with plenty of water for 15 minutes Skin Wash with plenty of water and soap. Cover irritated skin with an emollient Ingestion Do not induce vomit, loosen tight clothing Inhalation rest in a well ventilated area Xylene Mod. Flammable Toxic Fire Eye irritation Skin Irritation Ingestion Inhalation Safety Storage cabinet room, store in a well ventilated area away from ignition Use PPE Seek medical attention Eyes flush with water for 15 minutes Skin Wash contact areas with soap and water, wash contaminated clothing Ingestion Do not induce vomit, loosen tight clothing Inhalation -move to fresh air away from exposure Other Hazards Blood borne pathogens present in fresh human tissue Pathogenic in frozen sections since other specimens are fixed ex: HIV, HBV or TB cannot be excluded Eye contact, Mouth contact or any other mucous membrane. Cut by glass slide Use PPE Prevent spraying with cryospray preventing splashing of organisms Use Class III Safety Cabinet example for lymph nodes In case of known exposure take vaccines Sharp Objects Skin cuts Can contain infectious agents such as HIV, TB, HPV Sharps such as glass slides, scalpel, knives (for cut up), blades can penetrate the skin. Use PPE Go to infectious control unit for immunization for tetanus titer Burns caused by improper handling of hot items Chemical and Heat burns Body contact Acidic or alkaline chemicals and heat can burn the skin increasing risk for infection Use PPE Handle with care and away from the body Use appropriate cream In case of fire use fire extinguishers and fire blankets as discussed in the following fire section. Seek medical. Electrical injury (burns, shock, or death) Electrocution Flammable Death Electric shock user, equipment and user can catch fire. In both cases the user can die Inspect wires and replaced hazardous cords Use fused adaptors, prevent long extension wires Remove water from near wire Use fire equipment (as discussed in the following section), Do not touch electrocuted users or machinery Call medical help Fire Flammable Body burns Handle with care and away from the body For small burns use appropriate cream, Remove burning clothing if possible, Use fire extinguishers In histology CO2 type B extinguisher is good for flammable liquids and safe for electrical equipment. A Foam spray type A extinguisher is good for textiles, paper and wood. A fire blanket can be used or even a fire hose. A map showing the fire exits is available in the lab in case of fire alarms and uncontrollable fire in the laboratory Chemical Spillages Toxic or non-toxic Flammable Possibly corrosive Fire Eye contact Skin contact Inhalation Ingestion Use PPE Place in a safe place whilst handling chemicals Store flammable chemicals away from source of ignition Seek Medical Attention Eyes flush with water for 15 minutes Skin Use cellulose pads and alcohol on the area affected. Inhalation move to fresh air away from exposure Ingestion Do not induce vomit, loosen tight clothing and seek medical attention Decontaminate work surfaces FOR OFFICIAL USE ONLY Authorised: Date: