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Bridge UnderGraduate Science (BUGS) Programbridge.usc.edu/bugs
HD-CTC Fluid Biopsy Research Work Flow
Bridge UnderGraduate Science (BUGS) Program
Niha Choudhury
Bridge UnderGraduate Science (BUGS) Programbridge.usc.edu/bugs
From Blood to HD-CTC Samples
• Step 1: Process Patient’s Blood Sample 1. Collect plasma2. Lyse the red blood cells 3. Plate nucleated cells and dry
RESULT: Plated White Blood Cells (WBC’s) and Circulating Tumor Cells (CTC’s)
Bridge UnderGraduate Science (BUGS) Programbridge.usc.edu/bugs
Computational Analysis and Identification
• Step 2: Stain the Slides with Antibodies1. Fixation of cells and permeabilization of cell membrane primary
and secondary antibodies + DAPI dye o Cytokeratin (CK) Antibodies for Epithelial Cellso CD45 and Alexa Antibodies for WBC’s o DAPI Dye for nucleus o Fourth Channel Antibodies specific to the type of cancer
2. Live cell medium for maintaining the cells • Step 3: Automated Digital Microscopy with Scanner at 10X
Initial Analysis of scanned images to determine cell type: o Morphs, Re-Image [Unknown], Small CK, No CK, False Positives
RESULT: Computational identification of CTC’s by means of fluorescent imaging that highlighted the antibodies
Bridge UnderGraduate Science (BUGS) Programbridge.usc.edu/bugs
High Definition Images
• Step 5: Relocation and Reimaging 1. Relocation of cells of interest on the 80i Microscope to account for
coordinate shifts from the scanner to the microscope 2. Reimaging of all cells of interest at 40X using Fluorescent
Channels to highlight the antibodies [creates signal] o DAPI channel for DAPI dye (blue) o TRITC channel for CK in Epitheleal Cells (red)o FITC channel for Fourth Channel Markers [white]o CY5 for CD45 ofWBCs (green)
RESULT: Final, composite 40X images for CTCs of interest
Bridge UnderGraduate Science (BUGS) Programbridge.usc.edu/bugs
CTC Isolation
• Step 6: Picking Use of high-definition images as well as relocation coordinates to
pick each individual CTCs by means of the picking scopeo Single cells in respective PCR tubes
RESULT: A series of PCR tubes per slide with only a single cell in each
Bridge UnderGraduate Science (BUGS) Programbridge.usc.edu/bugs
Single Cell Genomics
• Step 6: DNA Amplification Series of polymerase chain reactions (PCR’s) to amplify the isolated cell’s
single copy of DNA • Step 7: Library Preparation
Collection of total DNA from single patient • Step 8: Genome-Wide CNV Profile
Analyze the CNV profile and heat map in order to reveal underlying genetic heterogeneity and clonal phylogeny around specific cancer types
ULTIMATE RESULT: Genomic data that would potentially reveal genomic patterns that could be used to evaluate drug effects or predict the possibility of relapsing when compared to other genomic data within a patient or across patients of
the same cancer type