Isolating highly enriched populations of circulating
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Transcript Isolating highly enriched populations of circulating
Isolating highly enriched
populations of circulating
epithelial cells and other rare
cells from blood using a magnetic
sweeper device
AmirAli H. Talasaz, Ashley A. Powell, David E. Huber,
James G. Berbee, Kyung-Ho Roh, Wong Yu, Wenzhong
Xiao, Mark M. Davis, R. Fabian Pease, Michael N.
Mindrinos, Stefanie S. Jeffrey, and Ronald W. Davis
Presented by: Casey Hua, Danyi Wu
Motivation
• Molecular Profiling Applications
• Improvement of:
• Cancer prognosis/treatment rare circulating
epithelial cells (CEpCs)
• Immune monitoring antigen-specific
lymphocytes
• Prenatal diagnosis fetal cells
Motivation
Problem: Current CEpC isolation platforms lack
purification efficiency
Solution: MagSweeper
–High capture rate
–High purity
–Enriches target cells (108x)
MagSweeper Process
1. Pre-label diluted blood samples with magnetic particles
2.
3.
4.
5. Repeat to eliminate background cells
improves purity at some cost to capture efficiency
MagSweeper Simulation
Magnetic flux
density
Magnetic forcedriven cell velocity
field
Cell capture
boundary
MagSweeper Simulation cont’d
Fluid
velocity/motion
Cell trapping profiles
Gap of uncaptured particles due
to fluid motion pushing
particles ahead of magnet
MagSweeper Performance
•
Enrichment Studies
1.
Rare Immunological Model Cells from a Mixed Sample (HLAA2)
•
•
2.
MCF7 Cancer Cells Spiked in Blood Samples
•
3.
Performance: isolating Circulating Epithelial Cells (CEpCs)
CEpCs from Breast Cancer Patients
•
•
Capture Efficiency
Purity
Application: CEpC isolation from Epithelial Cancers patients
Perturbation of Cellular Gene Expression
(1) Rare HLA-A2+ cells in Mixed Samples
Enrichment by ~2.5x105
(2) MCF7 Cancer Cells Spiked in
Blood Samples
Combining MagSweeper enrichment with visual inspection and
extraction of cells based on size:
•increase cell purification of the MCF7 cells from 51% to 100% purity
•similar cell capture rates from 62%± 7% of 59%± 27%
MCF7 cells isolated by MagSweeper with brightfield and fluorescence imaging.
(3) CEpCs in Breast Cancer Patients
•Found CEpCs in 47 of 47 samples from
patients with metastatic disease
•12±23 CEpCs per 9mL blood sample
MagSweeper isolation protocol does
not perturb gene expression
minimal changes in cell profiles:
94% of probe sets have changes
<50%
Fig. S2. Fold changes in logarithmic scale between the gene expression levels of
cultured cells before and after MagSweeper protocol.
Advantages of MagSweeper
• High enrichment
• No prior processing of blood required
• Flexibility in starting sample volume & process
scalability
• Further Optimization:
– Extension of the sweep to capture remaining particles in
gap left by fluid motion
– Refine magnet shape to yield more uniform magnetic field
gradient
Significance
• Diagnose malignancy from blood
– Improved cancer prognosis: CEpC numbers correlate better with
cancer progression than imaging, lymph node biopsy, and serum
biomarker analysis
• Enrich CEpCs for molecular analysis
– Identify surrogate endpoints for evaluation in clinical trials
• Extract individual CEpCs
• characterization of single cells for subpopulation studies on
heterogenous CEpCs