Lymphoma Diagnostics Techniques - Essay Example

LYMPHOMA DIAGNOSTICS TECHNIQUES al Affiliation) Problem of Interest Lymphoma Diagnostics. Majority of people die of a disease called non-Hodgkin’s lymphomas. The most common type of non-Hodgkin’s lymphomas (NHL), out of the many subtypes is the diffuse large B-cell lymphoma (DLBCL). Why the Problem Exists It is very difficult to categorize prognostic ally on morphological or clinical grounds the condition of NHL subtype DLBCL. With the complexity of the classification system of the NHL, it is widely known that the NHL categories have multiple disease subtypes each with separate molecular defects and clinical outcomes. For instance, 35-40% of patients diagnosed with DLBCL can cure with the use of chemotherapy whereas 60-65% of them die of the disease (Airley, 2009). Attempts to distinguish the patients who are likely to respond to chemotherapy from those unlikely have been unsuccessful. The DLBCL tumors are also not able to be distinguished using classical histopathology and the available molecular markers (Youngest & Coiffier, 2013). Summary of the project Recent studies on non-Hodgkin’s lymphomas show some diagnostic advances made possible by HD (High Density) DNA microarrays (Berar, 2003). Researchers currently have tried to approach the problems by using HD cDNA microarrays to profile gene expression in DLBCL. CDNA is a platform where Polymerase chain reaction products are separately and placed on the array surface using a robotic arrayed. The researchers identified genes responsible for classifying DLBCL specimens into separate subclasses. One of the subclasses was germinal-center B-cell-like DLBCL that showed an expression pattern the same as the mature germinal-center B-cells and portended a more favorable prognosis with a 60% five-year survival rate. Besides that, two other DLBCL subclasses, these are, type-3 DLBCL and activated B-cell-like DLBCL, presented much less favorable prognoses with a 35% five-year survival rate. From all these and the researches, it may be possible to determine the patients who are likely to benefit from therapies such as chemotherapy regimens or bone marrow transplants when DLBCL diagnoses. HD DNA microarray studies have highlighted molecular pathways that are important in cancer subclasses. Example, researchers have found out that several genes in the activated B-cell subclass of DLBCL were downstream targets of the NFkB transcription factor. They proved that NFkB activity is higher in this DLBCL subtype thereby suggesting that drugs targeting the NFkB may be effective in the treatment of these tumors (Cain, 2011). Gene expression profiling of tumor specimens may be useful in the pre-selection of patients who may benefit from drug treatment. It can use in examining gene expression profiles of cancers following chemotherapy to determine whether the tumors are responding to treatment. In this method, detailed patient-specific molecular information would use to predict an effective therapy (Schwab, 2009). Introduction DNA, key protein, or RNA markers to characterize disease are typically analyzed using molecular diagnostic tests. Molecular techniques, which uses effectively in clinical diagnostic laboratories, include Immunohistochemistry, DNA sequencing, PCR, western blotting, northern blotting, southern blotting and qRT-PCR. In these techniques, broadly trained technologists use to perform the tests rather than researchers who have specific methodological expertise. However, to allow maximum translation of post genomic research to patient care will require more parallel molecular tests which involve simultaneous analysis of tens to hundreds markers within a clinical sample (Nuber, 2005). Most of the basic research projects have switched their focus to methods to measure behavior of about 30,000 genes in the human genome. This research carries out at different levels within the gene, and measuring both phenotypic and genotypic changes. Parallel analysis at the DNA level, for example, it is applicable for analyzing sequence polymorphisms, which distinguish microbial genes, or human alleles that closely relates, at the level of DNA methylation patterns, at the level of mRNA to analyze patterns of gene expression and at the protein level. The development of microarray-based test used in diagnosing a broad range of diseases has led to the emergence of the ability to gain more information from a small clinical sample with parallel at expression analysis techniques. The greatest potential for the technology exists for the diseases that have a difficulty in histological differentiation, and serious consequences of inappropriate treatment. In the research environment, the analysis of gene products with microarrays has shown to provide effective diagnoses rather than analysis of individual proteins or RNAs. However, the continued development of new second-generation custom microarrays will be required in improving high throughput diagnostic methods (Berrar, 2003). Justification HD DNA microarrays have been very useful in semi-quantitative or comparative analysis of many (thousands) individual mRNA species. HD DNA microarray experiments suggest that it will be currently possible and even better to characterize disease states using new tests to measure the expression of tens to hundreds of genes, than the current method of using histopathology and the other molecular methods existing. The clinical application of this research basing on DNA microarray will demand further development of platforms for a refined new second-generation custom DNA microarray, which are most suitable for routine clinical laboratory testing (Li & Li, 2008). HD DNA microarrays are small devices, which have the ability to hold tens of thousands of gene capture probes on a solid surface at known positions. Among the most widely used platforms are the arrays by Affymetrix Inc. in Santa Clara, CA. This is a platform where the DNA oligonucleotides synthesize chemically, in situ, using masked photolithography on the array surface. Another platform that is also widely used is the cDNA array in which polymerase chain reaction products are separately generated and placed on the surface of the array with use of a robotic arrayed. These two platforms have been used by thousands of laboratories to catalog large numbers of gene expression in normal and diseased cells and tissues (Nuber, 2005). During the transition from normal to diseased state, tissues and cells undergo variety of biochemical and histological changes. These changes, in a large part, reflect the deregulation in the expression of genes. The normal and abnormal functions of cells are determined by the types and quantities of proteins, which are expressed from their respective genes. The DNA sequences of genes are first copied into mRNAs or other temporary molecules when they are expressed. The mRNAs are then translated into proteins. These proteins perform the functions and generate structures, which allow cells to conduct their biological and physiological activities. Researchers can take the advantage of the genetic flow of information, where it gets from DNA to mRNA to proteins, by purifying all mRNA populations expressed in the specimen, thereby synthesizing fluorescently labeled cDNA copies of the mRNA in vitro, and hybridizing to a microarray, the labeled cDNA. The level of mRNA expressed from the gene in the specimen is reflected by the intensity of the fluorescent signal at each gene specific probe on the microarray. Researchers are able to infer information concerning the quantities of gene expression and the types of proteins present, the state, and the biological behavior of the sample by measuring the quantities and types of tens of thousands of individual mRNAs expressed in the samples. For a very long time now, there have been alternative methods of identifying genes whose expression patterns are altered in disease. Even though this was possible, HD DNA microarrays really increased the productivity of research scientists in their efforts of gene research. The HD DNA microarray technology has increased the number of genes that can be identified within a unit time unlike the previous method, and has cut a lot of cost that was used in such processes (Simon, 2003). Preliminary protocol The initiation of carcinogenesis is generally by a genetic lesion, which results from an error occurring in the process of normal cell function or from unrepaired chemical or physical damage to the genome. In rare cases, the abnormal gene is inherited. In this event, there is a resultant increase in susceptibility to cancer for the family members who have inherited the gene. This event provides an increased possibility for development of additional genetic lesion, usually over several years. The cell will have full potential for generation of a malignant tumor when it acquires the proper combination of genetic lesions. Additional genetic alterations may be acquired, as the neoplastic cells go on dividing and expanding, and these, may contribute the characteristics, which make the tumor more clinically aggressive or resistant to treatment, such as resistance to death inducing signals and enhanced growth rate independence of growth signals. We can therefore say that the unique set of genetic lesions determine the characteristics and clinical behavior of a tumor. The pattern of mRNA expression in the cell is altered by this genetic lesion. The altered pattern can be regarded as the “fingerprint” of the tumor, or the “molecular signature” of the tumor. Tumors will have the very similar “signature” if they have genetic lesions, which are closely related. They will also have similar clinical behavior. Therefore, gene expression profiling will help a clinically relevant lymphoma classification, gene expression profiles will help in prognostication and personalized treatment, and make it possible to identify genes, which are important determinants of the behavior of lymphomas. Preparing of a cDNA library from extracted mRNA and performing massive sequencing of clones is one method of obtaining the gene expression profile of a tumor. This approach is not practical for profiling a large series of tumors, but is useful for gene discovery. A more efficient method, which still involves a substantial amount of DNA sequencing for each specimen and a complicated series of experimental manipulations, has been developed. This is the SAGE (Serial Analysis of Gene Expression) (Lee, 2004). The DNA microarray is principally a reverse of the Northern blot, where the various mRNA species “probes” are immobilized on a solid support, and the labeling and hybridization of the sample to be examined is done, to the immobilized probes. On each probe, the intensity of the hybridization signal is related to the corresponding mRNA concentration in the sample. There are usually thousands of immobilized probes on a DNA microarray, commonly on a solid non-porous support, even though some investigators still use membrane-based arrays. There are approximately 30,000 human genes, but there are more mRNA species, which arise through mechanisms like alternative splicing and others. A substantial proportional of all mRNA transcripts constitute a current collection of above 40,000 human cDNA clones. An array of 10,000 clones will be able to monitor the expression of major population of genes, especially when enriched with known genes and expression sequence tags of interest for a particular investigation, while most investigators do not use all of these clones in their experiments (Lee, 2004). Protocol for optimization There are two platforms of DNA microarrays, which are commonly used. In one platform, the spots on the microarray consist of PCR amplified products of cDNA inserts in plasmid clones. After necessary preparations, the polymerase chain reaction products are spotted on a poly L-lysine or aminosilane arrayed coated glass slide. The second platform contains oligonucleotide probes. The one manufactured by Affymetrix Inc., oligonucleotides are synthesized by the process of photolithography, in situ. There is also a possibility of synthesizing oligonucleotide probes off chip and then attached on microarrays (Nuber, 2005). The cDNA microarray has an advantage of flexibility of design. The flexibility of design allows more ready customization of the array to fit the needs of the investigators. In the event that a large number of arrays are required, it will be economical to produce them at an array facility other than purchasing commercial arrays. Apart from that, the fabrication of cDNA arrays and the quality controls are somewhat variable among laboratories. For example, the spots on the array may not be uniform and may vary significantly between and within arrays even for cDNA microarrays produced in the same facility (Simon, 2003). Hybridization is therefore performed typically, with the addition of a standard RNA preparation on the test RNA. The test RNA sample and the standard RNA sample are labeled with different fluorescent dyes by reverse transcription. The test versus standard cDNA hybridized ratio to each of the spots depends on their relative concentration in the mixture. The quantitation and expression as a ratio is done to the fluorescence due to the test and standard cDNA on each spot. The concentration of every cDNA can now be compared with each other since all the measurements are expressed as a ratio of the standard (Li & Li, 2008). Problems Associated With Proposed Solutions This method is not effective in very remote areas with limited facilities. The use of this technology causes many health risks, which may be caused by inhalation, skin contact, eye contact and poor disposal. Pollution is also another major risk, which is likely to be caused by poor disposal methods. There are also errors, which may occur due to poor handling and wrong procedures for dealing with the equipment. Approaches to troubleshooting Poor quality printing This can be because of poor printing environment, mostly inadequate humidity. Microarray showing high background after processing Shelf life and proper handling of the buffer should be ensured. No sample should be left to dry on the microarray surface while transferring the slides to wash stations. The microarray should always be washed after the processing Microarray showing high background after hybridization Impure cDNA was used for spotting, incorrect hybridization temperature, sample was dried on the microarray during hybridization reaction or the unincorporated dyes were not sufficiently purified away from the probe. No signal Defective cDNA microarrays or poor PCR amplification, probes washed away or poor probe labeling because of inefficient sample labelling. COSSH and risk assessment One of the major risks is the chemical output. The following first aid measures should be carried out. In case of eye contact, the eyes should be immediately flushed with plenty of water for 15 minutes. Medical attention should be sought if adverse health effects persist or become severe. In case of skin contact, the affected skin should be flushed with plenty of water. Remove any contaminated shoes and clothing. The clothes should be washed well before reuse. Shoes should also be cleaned thoroughly before reuse. If adverse healths effects persist of become severe, medical attention should be sought. In case any gases are inhaled, remove to fresh air. Oxygen should be given if breathing is difficult, and if not breathing, artificial inhalation should be given. If adverse health effects persist or become severe, medical attention should be sought. Spilled material and run off should not be disposed carelessly or made in contact with soil, drains, waterways and sewers. Appropriate disposal methods should be employed, where Safety eyewear complying with the approved standards should be used. Chemical resistant protective gloves and clothing should be used for skin protection A particulate filter respirator, which is properly fitted complying with an approved standard, should be used. Additionally, hands, forearms and face should be washed thoroughly after handling the chemical products, before eating, smoking or even using the lavatories, and at the end of the working period. Possibility of developing the application as a kit There is possibility of developing the application as a kit if more research is done in this line. A chip that resembles a glass microscope slide is currently used as a kit. Copies of the DNA of genes, which you wish to test for, are attached on the chip. DNA and genes are microscopic and therefore, the chip can contain one, or even thousands of genes, and remain to be very small. Genes, which are placed in this chip, are placed in known and specific locations. Colored fluorescent tags are then attached to the samples of cells, which are to be tasted. Healthy normal cells in many cases are included with different colored fluorescent tags. The sample cells are then mixed with the microarray chip. The DNA samples will then hybridize to the sites on the chip, which have the same genes as they do. When a laser is aimed on the chip, it will cause the lighting up of the sample cells. When examining where the sample cells light up, it will be possible to know the genes that they attached. From the above, it is possible to know the genes that the sample cells are expressing. When healthy cells are also included in the sample, both colors will be visible if they show up. Only health cells color can also shows up. In the event of the above colors, you know that sample cells, which are diseased, are under expressing certain genes, which are usually expressed by normal cells. This makes it possible to differentiate healthy cells from diseased cells, and to find out which genes are expressed on diseased cells and which are not. Looking at the current technology, it is possible to improve and come up with a kit that can use the HD DNA Microarray technology in diagnostics of lymphoma. Overcoming the Problems in a Systematic Ways Equipping the health centers with the necessary equipment that can be used to improve the human health status will help to overcome the problem of inaccessibility in remote areas. Installation of electricity and supply of DNA microarray biochips will be a key step towards overcoming problems associated with lack of the required facilities. Health effects can be minimized by the implementation of COSHH (Control of Substances Hazardous to Health, and through risk assessment. Regulations governing the disposal of related materials should be clearly stated and followed to the latter. Protective clothing and wears should be used when carrying out researches and experiments in this field. In cases of risk emergencies, the appropriate first aid measures should be employed. Consequently, In case any gases are inhaled, remove to fresh air. Oxygen should be given if breathing is difficult, and if not breathing, artificial inhalation should be given. If adverse health effects persist or become severe, medical attention should be sought. Spilled material and run off should not be disposed carelessly or made in contact with soil, drains, waterways and sewers. Appropriate disposal methods should be employed, where Safety eyewear complying with the approved standards should be used. Effects that come with errors can be corrected through trouble shooting techniques, which are given above. The general best functioning of the system requires following of procedures and all steps required and stated. REFERENCES Airley, R. 2009. Cancer chemotherapy. Chichester, West Sussex: J. Wiley. Berrar, D. P. 2003. A practical approach to microarray data analysis. Boston, MA: Kluwer Academic Publishers. Cain, H. J. 2011. A study of transcription factors STAT3, SP1 and NFkB in breast cancer. Newcastle upon Tyne: University of Newcastle upon Tyne. Lee, M. 2004. Analysis of microarray gene expression data. Boston: Kluwer Academic. Li, S., & Li, D. 2008. DNA microarray technology and data analysis in cancer research. Singapore: World Scientific. Nuber, U. A. 2005. DNA microarrays. New York: Taylor & Francis Group. Schwab, M. 2009. Encyclopedia of cancer (2nd Ed.). New York: Springer. Simon, R. M. 2003. Design and analysis of DNA microarray investigations. New York: Springer. Younes, A., & Coiffier, B. 2013. Lymphoma Diagnosis and Treatment. Dordrecht: Springer. Read More