3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The role of the polymerase chain reaction in the routine haematology laboratory.(1993) Gunther, Karen ElizabethThe Polymerase Chain Reaction (PCR) provides a means of amplifying target sequences of DNA exponentially and it is rapidly becoming an indispensable tool in the research laboratory. Many other molecular genetic techniques used for research are far too laborious and expensive to be used for routine diagnostic purposes but PCR has the potential to be different. This Research Report assesses the possible role of PCR as a routine diagnostic tool in the haematology laboratory. In the context of haematology, PCR can be used to detect both "pathological" and "physiological" target sequences present within the genome. Pathological sequences of interest would include mutations, deletions, insertions or translocations not present within the normal genome but which may arise either as a result of an hereditary abnormality or be acquired somatically. Sensitive detection of such sequences is useful for diagnostic purposes and can also be relevant in determining prognosis, evaluating response to therapy and following up minimal residual disease in the context of haematological malignancies. PCR detectable physiological sequences would include the immunoglobulin and T cell receptor gene rearrangements normally present within the genome of cells of the appropriate lineage. These rearrangements differ for each lymphocyte within a polyclonal population but are identical among members of a clone arising by proliferation of a single precursor cell. They can therefore be of value not only in determining cell lineage but also as markers of clonality. In this study the practical aspects of using PCR were assessed by setting up the technique of amplification of immunoglobulin gene rearrangements. The cost of reagents and disposable equipment, as well as that of major items of equipment required which are not usually available in a routine laboratory was also determined. In addition, peripheral blood and bone marrow samples reaching the Haematology Laboratory of the Johannesburg Hospital were analysed to assess the potential demand for such investigations. Once appropriate reaction conditions for the primers used had been established, PCR was found to be quick, technically simple and relatively inexpensive. Sufficient numbers of appropriate samples for which PCR analysis could potentially be of value were received in the Johannesburg Hospital Haematology Laboratory in the periods assessed, to indicate that diagnostic PCR, if available, would be well utilised. Some problems were encountered, particularly with regard to variability in the extent of amplification obtained. Thus for routine diagnostic purposes, extensive research and development of each set of primers to be employed will be necessary to make the technique more reliable and consistent. Adequate quality control will also be essential if PCR is to be used for diagnostic purposes. However, once these issues have been addressed, PCR should definitely find a place as a routine diagnostic tool in the haematology laboratory