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Title: An investigation into the value of molecular techniques in human lymphoid malignancies.
Authors: Akhtar, Naim.
Award date: 1989
Presented at: University of Leicester
Abstract: The value of molecular techniques in providing diagnostic clarification in human lymphoid neoplasms (malignant lymphomas), was investigated in comparison to conventional methods. Gene probe analysis (Southern blotting) and immunophenotyping were compared on biopsied tissues and peripheral blood of patients suspected of lymphoma. In the vast majority of B-cell lymphoma cases, gene rearrangement studies did not provide any additional clinically relevant information, but merely confirmed what was apparent from less costly and time consuming methods. Cases in which gene studies were required were uncommon, but included the heterogeneous and ill-defined T-cell lymphoproliferative disorders. As there is a lack of a specific phenotypic marker of malignancy in these T- cell neoplasms, gene probing provided the only definitive investigation of demonstrating clonal restrictions, and was found to be successful even on peripheral blood in lymphopenic patients. The technique of in-situ hybridization was used to differentiate monoclonal from polyclonal B-cell neoplasms, and was applied to routine bone marrow trephines to permit identification of light chain mRNA in suspected myeloma patients. In situ hybridization was found to be rapid, sensitive and specific for demonstration of light chain mRNA expression in plasma cell malignancies. The hybridization signal was optimised by an acid fixation and decalcification of the trephine, and by varying Proteinase K concentration. Cell morphology was well preserved and the resolution of this technique was high, with few problems of nonspecific stain uptake seen in immunocytochemical conventional methods. In situ hybridization can be modified to detect any target cellular nucleic acid sequence, after synthesis of an appropriate probe. Finally, the novel and powerful amplification technique of plymerase chain reaction (PCR) was investigated in B-cell lymphoma cases. Consistent and reproducible amplification was critically dependent on many variables including the assay conditions, the quality of Taq polymerase enzyme and the hybridization times and temperatures. However, false negative and of more concern false positive reactions still occurred. The exquisite sensitivity of PCR may not always be an advantage in clinical situations, and amplification of trace amounts of contaminants may become problematical. To be successful, PCR requires scrupulous technique and rigourous controls. PCR was shown to be potentially highly sensitive, and specific at distinguishing clonal from polyclonal B-cells and in detecting occult or minimal residual disease.
Type: Thesis
Level: Doctoral
Qualification: M.D.
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Health Sciences
Leicester Theses

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