This document summarizes the development of an automated filter-based system for isolating and detecting circulating tumor cells (CTCs) from blood samples. The system uses size- and deformability-based filtration to recover over 90% of CTCs while depleting over 99.99% of white blood cells. It also allows identification of protein markers on CTCs through immunostaining and detection of as few as 5 CTCs through quantitative RT-PCR analysis. Optimal sample storage conditions of 23°C for up to 72 hours was also determined.

1. Development of circulating tumor cell (CTC)
isolation and characterization
Anthony H. Tsai, Tatsuya Matsunaga, Nancy Vi
Hitachi Chemical Co. America, Ltd. R&D center, Irvine, CA
3) Remove magnetic
beads by column
filtration.
1) Add magnetic
beads to the blood-
cell mix.
2) Incubate on a
rotator at RT for
20 min.
Material and Methods
1) Dispense blood
into 3 mL aliquots.
2) Spike cells into
blood samples.
2) Process samples in the
Hitachi Chemical filter-
based system
1) Add blood-cell mix into
reservoir. Or add blood
into reservoir and spike
cells directly.
1) Scan cartridges with a
fluorescence microscope.
2) Image CTCs and WBCs.
Abstract
Circulating tumor cells (CTCs) are well recognized as a potential biomarker for cancer diagnosis. We have developed a highly efficient automated filter-based system to isolate and detect
CTC cells based on their size and deformability. The high cell recovery rate (90% or higher) and efficient white blood cells depletion (99.99% or higher) will be demonstrated by using
immunostaining method. The optimal storage condition for samples requiring long-range transportation will be addressed as well. In addition to the conventional three color staining, we have
developed a protocol that allows us to identify an extra protein (HER2) expressed on the CTC. Finally, the RT-qPCR result shows that our technology is highly sensitive (as low as 5 spiked cancer
cells in 3 mL of human whole blood) through molecular analysis.
The presence and amount of CTCs is well known as a prognostic biomarker, and has the
potential to be an indicator to monitor therapy response and predict drug efficiency1-2. CTCs can
be collected by a noninvasive blood collection method, and this enables multi-sampling for the
continuous tracking of therapeutic response. However, CTCs are rarer compared with the
common blood cells, such as platelets, red blood cells, and white blood cells (WBCs).
An automated filter-based system we developed is able to deplete the majority of those
common cells and retain the CTCs for further analysis including enumeration with
immunostaining and molecular analysis such as gene expression. The filter pore size of our
system can be precisely controlled by photolithography and the metal plating technology. Due to
the fact that cancer cells are isolated solely based on their size and deformability, not their
surface markers, our filter system can be applied to a broad range of cancers unlike an antigen-
antibody based system (e.g., EpCAM-based).
Results
1. CTC recovery rate
Molecular Analysis
1) Cell lysing
2) RNA extraction
3) cDNA synthesis
4) RT-qPCR
Donors
Runs
Average CV (%)
1st 2nd 3rd
1 112% 89% 79% 93% 18.1
2 91% 100% 106% 99% 7.6
3 103% 85% 93% 94% 9.6
@RT for 2 hrs, 24 hrs,
48 hrs, and 72 hrs.
3-color staining reagents 4-color staining reagents
Conclusion
1. Hitachi Chemical filter-based system is able to recover >90% CTCs and
deplete >99.99% WBCs from human whole blood after processing.
2. The optimal sample storage condition for our application is 23 °C.
3. A cancer related protein (HER2) immunostaining has been developed as the
4th color compared with conventional 3 color staining on the same image
4. RT-qPCR result demonstrates our technology is highly sensitive (as low as 5
spiked cancer cells in 3 mL whole blood)
Fig 1. Experimental Procedure
Table 1. Cell recovery rate of spiked 100 NCI-H358 lung cancer cells in 3mL human whole blood after processing
through Hitachi Chemical filter-based system
Introduction
1. Cristofanilli, M. et al., J. Clin. Oncol. 23, 1420-30, 2005
2. Pestrin, M. et al., Breast Cancer Res. Treat. 118(3), 523-30, 2009
3. Additional protein expression (HER2)
Fig 3. Establishment of HER2 expression detection on CTCs
Breast cells with different HER2 expression level were spiked into healthy donor blood.
Green arrow: Cell line, Red arrow: WBC, Scale bar, 50 µm
4. RT-qPCR
Fig 4. Molecular analysis of HER2 positive (SKBR 3) and HER2 negative (MDA-MB-231) spiked cells in human
whole blood after processing through Hitachi chemical filter-based system
dCt(HER2)= CtHER2-CtGAPDH (GAPDH: housekeeping gene)
2. Optimal sample storage condition
Fig. 2 (a) cell recovery rate of 100 spiked SKBR3 breast cancer cells (b) remaining WBC on the
filter after processing 3 mL of whole blood through Hitachi chemical filter-based system
(a) (b)
February 19-24, 2017
San Francisco, CA
© Hitachi Chemical Co. America, Ltd. 2017. All rights reserved