OPTIMIZATION OF FLOW CYTOMETRIC SORTING IN THE HEMATOPATHOLOGY LABORATORY

Abstract

University of Washington Abstract OPTIMIZATION OF FLOW CYTOMETRIC SORTING IN THE HEMATOPATHOLOGY LABORATORY Anju Thomas Chair of Supervisory Committee: Dr. Jonathan Fromm MD, PhD Department of Laboratory Medicine A subset of neoplasms in Hematopathology show rare neoplastic cells in a background of abundant non-neoplastic reactive cells. Developing a method of enriching for neoplastic cells, in in combination with efficient DNA extraction for molecular applications, could be a valuable tool for evaluation of the rare cell neoplasm (RCN). The purpose of this study is to optimize the procedures for flow cytometric cell sorting, affording enrichment of neoplastic cells of RCN. This project also addressed validation of an effective and automated DNA extraction method using minimal numbers of sorted cells. The detection level limit for clonality based molecular assay was also established used abnormal and normal sorted cells. In this study, we considered different technical aspects of the flow sorting such as how nozzle sizes will affect recovery of the various sorted cells. For this experiment, we used a megakaryocytic lineage induced cell ine, a Hodgkin lymphoma cell line (L428), and normal T lymphocytes. Another technical consideration evaluated, compared different precision modes using various percentage of the spiked cell to calculate the efficiency of sorting. Finally, these studies used sorted cells for comparison of two DNA extraction technique to determine which method afforded a greater yield of DNA for subsequent molecular studies; the B cell clonality assay platform was used for sorting abnormal and normal cells to establish detection levels. The studies suggest that it is necessary to have larger nozzle sizes for rare cell neoplastic sorting if large neoplastic cells from RCN. The precision modes study demonstrated that the combination of yield mode and purity mode is the preferable method for rare cells sorting. Comparison of DNA extraction methods proved that the EZ viral kit methods are more effective and sensitive when using the sorted cells than other DNA extraction methodologies; the limit of detection with B cell clonality assay determined that the level of detection using sorted cells could be hundred fold better than with unsorted bulk specimen. These studies demonstrated that by combining a reliable enrichment method with an effectve DNA extraction (for downstream molecular techniques) would improve the diagnostic value of these tests.