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Classification Of Canine Lymphoma On Human Working Formulation And Lymphocyte Immunophenotyping

University of Western Sydney

School of Science and Health

Unit Details

Unit Code:
401266
Unit Name:
Experimental Design and Analysis PG A
Credit Points:
20
Unit Level:
7
Assumed Knowledge:
Not Applicable

2.4 Assessment Details
2.4.1 Project report written in the form of a research publication

Rationale

Scientific writing is essential for any high-level research activity. This assessment aims at improving your scien- tific literacy in terms of scientific writing, data analysis and a critical appraisal of the published literature and the place of your work in the published literature. Successful completion of this assessment is a key skill in your training as a research scientist, or for the analysis of complex data in another field of employment.

Task

Students are required to report the outcomes of the research project in the form of a scientific research paper. The format of the paper will follow the

Answer

1. Introduction

Canine lymphoma is a sub-type of non-Hodgkin’s lymphoma (NHL) prevalent among humans and varies geographically. Canine lymphoma is denoted as the common type of cancer in the bodies of people as well as dogs. Lymphoma is the group of cancer, which stems from lymphocytes. There are three types of lymphoma, which are most common in dogs; those are Alimentary lymphoma, multicentric lymphoma, extranodal lymphoma, and mediastinal lymphoma. However, canine lymphoma is approximately similar to the non-Hodgkin's lymphomas in the human bodies. Chemotherapy is used in order to treat the spreading of lymphoma in the patients. Proper understanding of the canine lymphoma can help the dog owners to relate and to treat the conditions of dogs. In order to diagnose the canine lymphoma in patients, the veterinarians gather samples of the affected organs. 

In this study, a retrospective study has been done among 120 dogs that are affected with canine lymphoma. In addition, a critical analysis of the literature review part helps the researcher in order to derive the research frameworks. In this study, immunological as well as cytohistological types of canine lymphoma along with the symptoms have been analysed briefly. Importance of p53 expression, PGP and the histological grading of canine lymphoma have been identified. Moreover, working functional methods of the canine lymphoma grading has been highlighted.

1.1 Background  

Canine lymphoma is very much common in dogs. In this disease, the lymph nodes of dogs would be enhanced by 3 to 10 times as compared to the natural sizes. This disease may appear in the bone marrow, skin, and eye and in the gastrointestinal tracts of the dogs (Teske, Wisman & Moore, 1994). Canine lymphoma can be denoted as the diverse groups of cancers that affect mostly the dogs and owners of dogs. From a recent study, it can be known that nearly 7 to 14% of the cancers are the canine lymphoma among every type of cancers found in dogs (purdue.edu, 2019). In addition, there are more than 30 types of canine lymphoma, which would vary according to the behaviours. As opined by Behling-Kelly et al. (2018), canine lymphoma would affect mostly the lymph nodes of the dogs, however, other organs like liver, bone marrow and spleen of the patients can be affected by canine lymphoma. 

The diagnosis of the canine lymphoma is mostly similar to the notion of Hodkinson's lymphomas (NHL) that may affect human bodies. As opined by Burton et al. (2018), both NHL and canine lymphoma give similar types of responses while examined microscopically and under the chemotherapy. From a recent report, it can be seen that NHL had been diagnosed among 65,000 people in Australia and it is a seventh most common type of cancer overall (purdue.edu, 2019). The exact reason behind the attack of this disease is not known yet. However, it can be said that the spreading of bacteria, viruses, and chemical exposure are the common trigger factors of canine lymphoma. As suggested by Carrasco et al. (2015), most common symptoms of canine lymphoma among digs are the enlarged, non-painful and firm lymph nodes. Some other symptoms of the disease are lethargy, losses of appetite, weight loss, and edema. The best ways for diagnosing the canine lymphoma is the biopsy.  

1.2 Problem statement

About 176 classes of canine lymphomas have been identified based on their morphological variations using four different human lymphoma classification schemes. The expression probability of p53 and p-glycoprotein (PgP) is responsible for the variations of the canine lymphomas. Hence, there creates an urge to identify and segregate them for pathological and therapeutic convenience using an appropriate identification and classification scheme (Dhaliwal et al. 2013). 

In the aspect of the canine lymphoma, no specific reason has been found behind this disease. In addition to it, no proper cure has been identified for reducing the spearding of the disease expect if the chemotherapy. Therefore, it is required to identify the proper reason behind canine lymphoma. It has been found that the NHL is spreading surprisingly in men. However, the NHL also affects the dogs. 82 NHL had been classified according to the morphology (according to Working Formulation) and the immunophenotypes (identified with the anti-T-cell as well as anti-B-cell antibodies). Hence, the problem is to identify the surprising relationship between the NHL and the canine lymphomas in the human bodies.  Moreover, in order to evaluate the effectiveness of human working formulations as well as immunophenotyping in classifications of canine lymphoma, the study has to be conducted by researcher.

1.3 Aim

The aim of this research is to evaluate the applicability of the human working formulation method to classify canine lymphoma.

1.4 Objectives

  • To identify the susceptible criteria within dog      population using a retrospective epidemiological method
  • To analyse the relevance of the applicability as      per past literatures on Immunophenotyping and work formulation
  • To evaluate the effectiveness of the human      working formulation and immunophenotyping in canine lymphoma classification
  • To recommend measures for implementing the work      formulation classification in pathological usage

1.5 Research questions

1.5.1 Primary question:

Does similar lifestyle and environmental factors affect the working formulation as lymphoma grading method benefiting prognosis and therapeutic treatment of canine lymphoma?

1.5.2 Secondary questions:

  1. What is the probability of occurrence of canine lymphoma      among the dog population?
  2. To what extent are the two methods relevant as      per past studies?
  3. How much effective are human working formulation      and immunophenotyping in canine lymphoma classification?
  4. In what manner can work formulation be      implemented for the above classification?

2. Literature review

2.1 Lymphoma and its classification

Lymphoma is the most common disease among animals, especially among dogs.  Kind of malignant tumours increase the risk of cancer among dogs. Canine lymphoma is similar to non-Hodgkin's lymphomas (NHL) that occurs in the human body. Most possible causes for canine lymphoma as bacteria, viruses, physical factors and so on. Lymphoma breaks down the immune system of the dogs, which cause cancer and affects a different part of the body. In the words of Carter et al. (1986), most of the lymphoma is found in the lymph nodes of the dogs. In this regard, it can say that lymphocytes are the main elements that affected by virus and bacteria’s that cause lymphoma in dogs. Lymphocytes play a vital role in the human body as well as in the animal body. In the words of Pawlak et al. (2016), it helps to fight against infection. During lymphoma white blood cell or lymphoma are affected by some virus that totally breaks the immune system of the body. It can say that immunophenotyping is used to cure such diseases. Flow cytometry are also used as a too, for the treatment of canine lymphomas.  

Lymphoma can be categorized in different ways. As mentioned by Behring-Kelly et al. (2018), lymphoma can be classified as low grade and high grade as per its intensity. The four types of lymphoma are meditational, gastrointestinal, extra nodal and multicentre lymphoma. Mediastinal lymphoma occurs in lymph nodes of the dogs. Furthermore, gastrointestinal occurs in the stomach as a form of a solitary tumour. As mentioned by Carrasco et al. (2015), multicentric lymphoma can be found liver, lymph nodes, bone marrow and so on. Additionally, this is the most common lymphoma among dogs. Extranodal occurs in the skin, eye, and health of animals. It can say that any type of lymphoma becomes malignant for the dogs. Most of the affected dogs cannot survive more than approx 215 days. For this reason, the treatment of canine lymphoma must be handled with care. 

Besides, lymphoma can be categorized into two subtypes such as B-Cell and T-Cell. As mentioned by Ohmura et al. (2017), these subtypes are dived into seven categories that include benign hyperplasia, low-grade T-cell, centroblastic large B-cell, low-grade B-cell, immunoblastic large B-cell, high-grade T-cell and B-cell and high-grade peripheral T-cell. High-grade lymphoma can create higher mortality rate and it affects badly inside the body. In this type, the immunity power of the animal can break down. On the other hand, low-grade lymphoma has a lower mortality rate than high-grade lymphoma. In high-grade lymphoma, affected fogs are ca not survive more than 210 days as it totally affected in the bone marrow of the dogs. Such a situation, canine lymphoma is not curable. In the words of Burton et al. (2018), a dog who is suffered from Low-grade T-Cell can survive more than high-grade T-cell lymphoma affected dogs. 

2.2 Importance of PGP, p53 expression and histologic grading in canine lymphoma pathological identification

P53, the tumour suppressor plays the most important role in terms of DNA repair as well as damage. As mentioned by Motegi et al. (2018), it can the cause of canine cancer and can effects the immune system badly. P53 expression is used for the resistance of canine cancer for dogs. In this context, sulphate is delivered to the dogs for the treatment of p53 expression in terms of canine lymphoma. As suggested by Pinello et al. (2017), it can observe that there are many genes, which can resist drug. In such a situation, a cytotoxic drug may fail in the process of chemotherapy. As a result, p53 genes are introduced to response in chemotherapy or in radiotherapy.  As suggested by Ponce et al. (2010), it can say that the condition of p53 gen must monitor before a diagnosis of canine lymphoma. P53 gene is consisted of nuclear phosphoproteins and has 393 amino acids to specify DNS sequences. In the words of Marconato et al. (2017), p53 gene can rest the growth of cancer among dogs. In this regard, such proteins can be implied to the body of the animal so that they can be protected from malignant lymphoma. 

Permeability glycoprotein or PGP is one of the common drug treatments to resist canine lymphoma among dogs. As stated by Miniscalco et al. (2018), it can be detected under lots of psychological conditions of a normal organ such as skin, eye, gland, bone marrow, lung, brain and so on. Most of the multicentric lymphoma is treated under PGP expression. As stated by Levi et al. (2016), the rate of positivity and intensity of Permeability glycoprotein determine under the observation of microscopic organism. Most of the multicentric canine lymphoma can be treated based on PGP and p53 protein. It can clear that this protein is the most important medicine to build immunity power during the treatment of lymphoma. 

Immunohistochemistry is associated with these two elements such as p53 and PGP. This process is performed to fix formalin and paraffin embedded. In this regard, it can say that the canine lymphoma affected dogs are treated under the protocol of chemotherapy. As mentioned by Greenlee et al. (1990), survival and remission duration can be evaluated through historical grades and the presence of important drugs in the market. PGP and p53 is the most significant expression in terms of the treatment of canine lymphoma. 

2.3 Immunological and Cytohistological variations of canine malignant lymphoma

A cytological examination is one of the most important diagnosis programs for canine lymphoma. By these methods, lymph nodes can be treated quickly and sensitively. It is helpful for diagnosing mainly low-grade lymphoma. Moreover, an excisional biopsy can be done to remove the lymph nodes permanently. In the words of Fournel-Fleury et al. (1997), a cytological expression can help to analyze the flows of fine-needle aspirates from the lymph of neoplastic, which decrease the importance of excisional biopsy for canine lymphoma.  In the words of Sayag et al. (2018), cytological variation shows the effects of high-grade lymphoma over the low-grade lymphoma among dogs. It also differentiates different types of lymphoma such as centroblastic lymphoma and polymorphic lymphoma. 

As per the World Health Organisation, lymphoma is classified into two categories such as low-grade lymphoma and high-grade lymphoma. In this regards, low-grade lymphoma is recorded as 24.5 % while high-grade lymphoma is recorded as 75.5%. Besides, clinical sings classify lymphoma as extranodal lymphoma and lymphadenopathy diseases. As mentioned by Teske et al. (1994), immunohistochemistry recorded 63.8% for B-Cell, 08 % for Null-Cell and 35.4% for T-Cell. Some low-grade lymphoma represents marginal zone lymphoma and follicular lymphoma. As suggested by Dhaliwal et al. (2013), it observes that marginal lymphoma is most common among animals whereas follicular lymphoma is rare in the case of canine lymphoma. On the other hand, high-grade lymphoma has sub types such as small B-cell lymphoma, plasmacytoid lymphoma, mediastinal anaplastic large B-cell lymphoma, and Burkitt-type B-cell lymphoma. T-cell lymphoma has some subtypes such as low-grade prolymphocytic T-cell lymphoma and aggressive granulocytic large-cell lymphoma. 

Immunophenotyping is performed on the basis on cytological biopsy test for canine lymphoma. In this process, some antibodies such as CD21, CD, 20, CD 3, CD8 and CD4 are used for high-grade T-cell lymphoma. T-cell neoplasia is revealed by the cytological analysis of canine lymphoma. In the words of De Arespacochaga et al. (2007), it can say that each of the types of lymphoma needs important medicine to cure the disease. The majority of lymphoma can appear as T-cell lymphoma among dogs, which are quite accurate malignant lymphoma and most of the time it is not curable. Such types of lymphoma cause death to animals. In this regard, it can say that no lymphoma can be expressed in terms of double phenotypes. 

2.4 Immunophenotyping as a method to classify canine non-Hodgkin's lymphoma

Immunophenotyping determines the antigen expression over particular cells or tissues by flow microscopic and cytometric detection. In the words of Zandvliet et al. (2015), cytometric approaches deal with large cell by detecting its issue and indices. On the other hand, microscopic approaches assess tissues architecture and cellular morphology for a specific tissue. As mentioned by Corner et al. (2017), lymphoma is mainly diagnosis under the canine hematopoietic system. Polymerase chain reaction (PCR) and cytometry are examined to study the canine lymphomas. Immunophenotyping label white blood cells or lymphocytes with antibodies against the surface proteins. The entire process can be performed form bone marrow, blood cells or within spine fluid. On the other hand, it can be done thorough flow cytometer. 

Immunophenotyping is specially used for the diagnosis of special types of cancer including lymphoma. This is done by comparing the affected cells with the normal cells in a living body, so that the antigens could be identified with help of the antibodies. The antibodies in this process detect the cells that are called markers, which are the specific antigens. The cancer affected cells in lymphoma that are the complex populations are identified and sorted in immunophenotyping using the blood sample for the treatment process of those specific cells. This technology is commonly used in normal research purposes as well as in the clinical laboratories for the treatment process. In this regards, the affected dogs are going under the process of immunophenotyping by using flow cytometry 

Such process is commonly used in flow cytometry. Flow cytometry of lymph nodes, blood and bone marrow can improve prognosis and evaluation of dogs that are suffered from lymphoma. As mentioned by Comazzi et al. (2017), it can say that such a system mainly used s research tool but not use wide sped in terms of lymphoma. In this context, they can observe that European laboratories provide such practices to veterinary patients. Immunophenotyping treatment can increase the survival time of dogs. A T-cell phenotype has lower survival time and shorter relapse-free time. Besides, B-cell type has a long survival type for affected dogs. 

2.5 Working function method of canine lymphoma grading

Lymphoma can be determined in different phases. Such disease must use immunophenotyping process to determine the root cause of the issues. In this regards, canine lymphomas need proper antibodies to fight against cancer cell. In this situation, fine-needle biopsies can be done through lymph nodes. Immunophenotyping uses Ki-67, CD3, and PAX5 for the treatment of lymphomas. In the words of Carrasco et al. (2015), lymphocytes are immunostained with monoclonal and fluorescent-labelled against CD21 and CD3. The effectiveness of immunophenotyping can be determined through flow cytometer by differentiating B and T cell immunophenotypes. As suggested by Zandvliet (2016), low-grade type follicular tumours are rare and not too much malignant. On the other hand, high-grade type aggressive tumours are most common for canine lymphoma. High-grade lymphoma is more malignant than low-grade lymphomas. 

There are 166 types of canine lymphomas, which are malignant can be classified hislotogically. Follicular lymphoma is a system of low-grade lymphoma that is growing slowly in the animal body. Approx 69.9% of lymphomas are considered as histolytic lymphomas. In the words of Vajdovich et al. (2018), it consists of different types of immunologic and morphologic tumours that cause malignant cancer. In the process immunophenotyping, 95 lymphomas perform as polyclonal and monoclonal antibodies. Low-intensity lymphoma is mainly B-cell types. Such types of lymphoma are not too much malignant. In the words of Comazzi et al. (2015), it can be cured by using some drug. Immunophenotyping concentrates on the nuclear size, growth pattern of neoplastic and nuclear morphology. It is a process of test, which determines the antibodies form B-lymphocytes and T lymphocytes. It can detect cancer for an animal. For this reason, immunophenotyping is most important for the treatment of lymphoma among dogs. 

Cytological analysis helps to identify the forms of lymphoma against antibodies. The pan-B anti-mb1 and pan-T anti-CD3 polyclonal antibody can be used in terms of lymphoma. Polyclonal and monoclonal antibodies are used for the determination of canine lymphomas. As stated by Chhieng et al. (2001), the working formulation also categorizes lymphoma into two categories such as polymorphic lymphoma, which has centroblasts components and micro nucleated medium-sized cells, which consist of small-cell variants. T-cell neoplasia is the appearance of follicular lymphomas and it can observe that micro nucleated medium-sized cells are originated in the zone of the prifollicular zone. 

3. Methodology   

3.1 Research design              

Research designs are characterized as three types, those are explanatory method, exploratory and descriptive research designs. In order to carry out social or academic the learners use types of research, above three methods. In exploratory type, psychological as well as mental perspectives of the participants need to consider. As opined by Abdulkadiroğlu et al. (2017), in explanatory type, learner always analyses gathered information and does not judge the negative effects of the theories. Moreover, in the descriptive, research study always possess pre-specified aims as well as objectives. The researcher in this method analyzes both of the aspects of theories and data.  

In this study, researcher selects Cohort studies, where subjects are gathered in a disease-free manner for examination purpose at the beginning of observation period. As opined by Lewis (2015), Cohort study would allow the examiner for calculating incidences of diseases within exposure groups. Through Cohort study, absolute risk of incidence, relative risks, and risk difference can be known. Retrospective epidemiological method will be used to have a descriptive study. The hypothesis will be analysed using a deductive approach. 

Justification for choosing Descriptive research design

This research study possesses pre-specified sets of aims and objectives. In this study, learner has selected Retrospective epidemiological method in order to create descriptive study. Descriptive design would assist the researcher to analyze the study from a larger population and samples within a short time period. Here, researcher has done the retrospective study among 120 dogs with canine lymphoma. Retrospective study examines the exposures towards suspected risk factors related to the outcome of the specific study. Errors due to bias and confounding are most common in the retrospective study. Due to this problem, the retrospective type of investigation is criticized often.

3.2 Data collection methods      

Researcher uses both primary and secondary method for collecting information during research investigations. This will involve both primary and secondary data collection. Primary data will be collected from 120 dogs affected with this form of lymphoma. Secondary data will be collected from clinical and pathological reports of patients suffering from this type of lymphoma. immunophenotyping and histological blocks of lymphoma cases will also be studied.

Primary Research

Primary researches are conducted often among the people who have enough sources of knowledge about the research topic. As opined by Abdulkadiroğlu et al. (2017), primary researchers take more time and cost more than secondary research. Few types of primary researches are focus groups, surveys, interviews, and questionnaires.  

Quantitative Method

The quantitative method comprises any of the methods that would result in numerical values. In the words of Tight (2016), quantitative data collection processes include clinical trials, surveys, experiences, and questionnaires. In this study, the researcher has conducted experiment by taking 120 canine lymphoma affected dogs. Learner has used epidemiological method and Cohort study. As suggested by Lewis (2015), in the cohort study, the outcome is determined after the exposure. In addition, this study is expensive and often takes longer time than the usual experiments. Hence, in order to find out probability of the occurrence of lymphoma among dogs, this method is opted by investigator.

Qualitative Method 

This method of data collection is also part of primary data gathering methods. As highlighted by Abdulkadiroğlu et al. (2017), focus groups and interviews are the common types of data collection, which are used within qualitative researches. In this study, learner has not chosen qualitative method for data collection.  On the other hand, as argued by Lewis (2015), secondary research is conducted via compiled, published and organized data of others. In this study, researcher opts for secondary data collection process in order to analyze the effectiveness of human working formulations within classifications of canine lymphoma.  

Hence, secondary information has been gathered from the pathological and clinical reports of the patients from canine lymphoma. Both of the dogs and owners of the dogs might be affected by this type of lymphoma. Along with it, in this research study, histological blocks and immunophenotyping tests of lymphoma cases have been studied. 

3.3 Methods of data analysis

Thematic analysis for secondary data will be included. The primary data will be analysed related to factors such as –sex, sixe, age, breed of the dog and location of the cancer. Qualitative and quantitative data analysis will be done for providing accurate and relevant data over these issues. As such, researcher conducting primary data focusing over 120 dogs who are getting affected through this disease. It will mainly shed light on a particular breed, age, sex or size and most important location or parts they are getting affected. On the other hand, collection of secondary data that is online journals, articles and histological Studies of the previous researchers over the topic helps to determine source and factors that have been already determined by them over these lymphomas. It also shows a different type of techniques that are used by the researcher to complete the research work over this topic. A previous journal article will provide knowledge about canine lymphoma and its effect over dogs that will be helpful for future research. This collection of data helps to determine the cause and provide accurate and relevant information over this lymphoma. In this regard, the online article will give relevance case study for the research. It will provide similarities between the research questions. Online case studies will help to understand the significance of the study about lymphoma. As a result, this data collection process helps to determine the accurate and relevant source to gather information and provide a relevant source for this researcher paper. 

3.4 Timeline














Month 1 2 3 4 5 6
             
             
 literature   review
 Histology  ImmunophenotypingData Analysis
  




 

 based on   Working Formulation
         
 
 

Table 1: Timeline

(Source: created by author)

3.5 Resource and budget for this project 

Some resources are required to meet the requirement of this research paper. Such as pathology reports on canine, lymphoma helps to identify the cause of lymphoma and understand the growth of lymph’s nodes within the body of the dog. Besides, such reports will also show the amount of affected areas that must be treated with some proper diagnosis. Clinical reports of the affected dogs will also helpful for the determination of the disease. Histology blocks will be more helpful for this research histology block will show the actual affected areas for canine lymphomas. Such a report will be ideal for lymphoma studies. Additionally, it will identify the immune system and structure of the body. Immunophenotyping antibodies will be needed for examining the rate of infection in the body of the specific dogs. It will ideal for differentiate the different stages of canine lymphoma by using antibodies. However, such research resources will be costly. In this regards, the estimated budget for antibodies will be $1000. Additionally, the budget for histology reagents and consumables will be $1000.



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