BUGS Work Flow Presentation

Bridge UnderGraduate Science (BUGS) Programbridge.usc.edu/bugs

HD-CTC Fluid Biopsy Research Work Flow

Bridge UnderGraduate Science (BUGS) Program

Niha Choudhury


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)


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


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


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


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

BUGS Work Flow Presentation

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