Genomic applications of laser capture microdissection

This thesis focuses on the use of laser capture microdissection (LCM) in genomic applications. It aims to demonstrate that a wide variety of gene specific and genome wide analyses are possible after LCM. The thesis is divided into two major parts. In a first part, the use of LCM in forensic research is discussed. The introduction summarizes all possible forensic applications of LCM, gives an overview of the staining and detection options and reviews the DNA extraction protocols compatible with LCM of cells from forensic samples. This overview is followed by an evaluation of three DNA isolation methods after LCM. To conclude the first part of this thesis, two chapters describe the use of LCM in sexual assault cases. The second part of this thesis focuses on the use of LCM in clinical research. Three different disorders have been studied to indicate that LCM can be used in a very broad range of clinical applications. The first chapter after the introduction describes the use of LCM to isolate proliferating B-cells from pseudofollicles in lymph node sections of patients with chronic lymphocytic leukemia. The aim of this study was to demonstrate a clonal relationship between these cells and the accumulating leukemia cells in the blood of these patients. In the second chapter, LCM is used to isolate single peripheral blood mononuclear cells from patients with a mitochondrial point mutation leading to a neuromuscular disorder. Using restriction fragment length polymorphism (RFLP) analysis, the mutation load was determined in these single cells, showing a high intercellular variability in heteroplasmy. In the final chapter, LCM was used to isolate metastasized neuroblastoma cells from bone marrow aspirates. Subsequently, array-based comparative genomic hybridization (arrayCGH) profiles from the metastasized cells and the primary tumour were compared to demonstrate that these metastases can be used for diagnosis and prognosis in cases where primary tumors are not available