ABSTRACT
Single-cell RNA sequencing (scRNAseq) is a rapidly advancing field enabling the characterisation of heterogeneous gene expression profiles within a population. The cell cycle phase is a major contributor to gene expression variance between cells and computational analysis tools have been developed to assign cell cycle phases to cells within scRNAseq datasets. Whilst these tools can be extremely useful, all have the drawback that they classify cells as only G1, S or G2/M. Existing discrete cell phase assignment tools are unable to differentiate between G2 and M and continuous-phase-assignment tools are unable to identify a region corresponding specifically to mitosis in a pseudo-timeline for continuous assignment along the cell cycle. In this study, bulk RNA sequencing was used to identify differentially expressed genes between mitotic and interphase cells isolated based on phospho-histone H3 expression using fluorescence-activated cell sorting. These gene lists were used to develop a methodology which can distinguish G2 and M phase cells in scRNAseq datasets. The phase assignment tools present in Seurat were modified to allow for cell cycle phase assignment of all stages of the cell cycle to identify a mitotic-specific cell population.
Subject(s)
G2 Phase , Mitosis , Mitosis/genetics , Humans , G2 Phase/genetics , Single-Cell Analysis/methods , Sequence Analysis, RNA/methods , Histones/metabolism , Histones/genetics , Gene Expression Profiling/methods , Computational Biology/methods , SoftwareABSTRACT
PURPOSE: The aim of this project was to design and manufacture a cost-effective end-to-end (E2E) phantom for quantifying the geometric and dosimetric accuracy of a linear accelerator based, multi-target single-isocenter (MTSI) frameless stereotactic radiosurgery (SRS) technique. METHOD: A perspex Multi-Plug device from a Sun Nuclear ArcCheck phantom (Sun Nuclear, Melbourne, FL) was enhanced to make it more applicable for MTSI SRS E2E testing. The following steps in the SRS chain were then analysed using the phantom: magnetic resonance imaging (MRI) distortion, planning computed tomography (CT) scan and MRI image registration accuracy, phantom setup accuracy using CBCT, dosimetric accuracy using ion chamber, planar film dose measurements and coincidence of linear accelerator mega-voltage (MV), and kilo-voltage (kV) isocenters using Winston-Lutz testing (WLT). RESULTS: The dedicated E2E phantom was able to successfully quantify the geometric and dosimetric accuracy of the MTSI SRS technique. MRI distortions were less than 0.5 mm, or half a voxel size. The average MRI-CT registration accuracy was 0.15 mm (±0.31 mm), 0.20 mm (±0.16 mm), and 0.39 mm (±0.11 mm) in the superior/inferior, left/right and, anterior/posterior directions, respectively. The phantom setup accuracy using CBCT was better than 0.2 mm and 0.1°. Point dose measurements were within 5% of the treatment planning system predicted dose. The comparison of planar film doses to the planning system dose distributions, performed using gamma analysis, resulted in pass rates greater than 97% for 3%/1 mm gamma criteria. Finally, off-axis WLT showed MV/kV coincidence to be within 1 mm for off-axis distances up to 60 mm. CONCLUSION: A novel, versatile and cost-effective phantom for comprehensive E2E testing of MTSI SRS treatments was developed, incorporating multiple detector types and fiducial markers. The phantom is capable of quantifying the accuracy of each step in the MTSI SRS planning and treatment process.
