| Summary: | A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Haematology. === Introduction
Genetic abnormalities play an integral role in the pathogenesis, diagnosis, prognosis and treatment of leukaemia. As the technology advances in the field of molecular testing, so will our approach to the diagnosis and treatment of patients presenting with suspected leukaemia. Our aim was to evaluate the Signature® LTx Multiplex RT-PCR assay which combines multiplex RT-PCR (Reverse Transcription Polymerase Chain Reaction) with multiplex fluorescent bead-array detection and to compare results with those of routine diagnostic tests FISH (Fluorescent In Situ Hybridisation) and cytogenetics analysis. The assay has 12 translocations commonly associated with acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML).
Material and Methods
RNA was extracted from bone marrow aspirate and peripheral blood samples from both adult and paediatric patients presenting with ALL, AML and CML and analysed 70 samples using the multiplex assay with four steps of reverse transcription, DNA amplification, hybridisation and detection. Qualitative analysis was performed with a pre-determined mean fluorescence intensity (MFI) cut off level as well as quantitative analysis of signal distributions for positive, negative and endogenous controls (GAPDH: glyceraldehyde 3-phosphate dehydrogenase). Results were then compared to those of cytogenetics and FISH.
Results
Eighty six percent of the samples had concordant results and 14% showed discordant results compared to routine diagnostic tests. Of these discordant samples, some results could be explained by suboptimal RNA quantity or GAPDH MFI signals and if these were repeated or
discarded, the number of discordant samples would decrease to four percent. The multiplex detected four translocations which were not identified by FISH or cytogenetics and 6 mutations were missed by the assay, however the majority of these are attributable to suboptimal RNA and internal control signals which would be repeated in the routine use of the assay.
Conclusion
Overall, the Signature® LTX Multiplex RT-PCR assay showed good correlation with gold standard test techniques. While the assay provides possible advantages including ease of use and improved turnaround times, drawbacks include problems with RNA quantity after extraction and logistical concerns including the need to batch samples for analysis which will affect the turnaround time. Careful cost analysis and possible modification of the translocations included in the assay would be required before the assay could be incorporated as part of the routine diagnostic work-up of patients presenting with leukaemia. === MT2016
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