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APPLICATION NOTE
Cytogenetic Monitoring of Cancer Progression in Circulating Tumor Cells
Utilizing the IsoFlux System and GenASIs Spectral Imaging Technology
Overview
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Circulating tumor cells (CTCs) have shown promise as a clinically-relevant sample for monitoring the genetic variation of cancer progression
Fluorescence in situ hybridization (FISH) is a single-cell approach to understanding patient-specific molecular profiles of disease
FISH analysis of CTCs offers a less invasive method for tracking disease progression at the genome level
The IsoFlux System (Fluxion Biosciences) provides a robust method for accessing CTCs and optimizing use in FISH assays
GenASIs (Applied Spectral Imaging) provides a cutting-edge hardware and software platform for high resolution imaging of FISH slides
Introduction
Fluorescence in situ hybridization (FISH) is a technique that elucidates chromosomal context of a specific DNA or
RNA sequence. A cellular sample is fixed onto a pathology slide and hybridized with one or more fluorescentlylabeled probes. Using a fluorescence microscope, the location and prevalence of the target sequence is visualized.
From these data, genomic aberrations can be determined, including gene amplification, translocation, and deletion,
all of which are important hallmarks of disease subtyping. FISH has emerged as a clinically-useful tool for understanding patient-specific traits of cancer at the molecular level. FISH is typically performed on biopsied tissue
samples obtained during surgery. These samples are generally available only at the time of diagnosis and subsequent samples are difficult to obtain. Circulating tumor cells (CTCs) have emerged as a potential source of
clinically-useful information that can be accessed with far greater frequency than tissue biopsies.
FISH using CTCs has many potential advantages as an analytical and diagnostic tool. The fundamental principles
of probe application are well developed at this point, and many labs already have the hardware and software
necessary to process FISH samples. Several therapeutically-actionable targets for FISH have already been
clinically validated such as Her2 and ALK, with many more in the development process. FISH provides single cell
data which can help elucidate the heterogeneity of the disease, such as the percentage of tumor cells which are
Her2+. Despite these advantages, FISH on CTCs is a non-trivial process and requires a sensitive CTC isolation
method and a high resolution imaging system.
Current Challenges for Running FISH on CTCs
Analyzing CTCs using FISH presents numerous challenges. Enough cancer cells must be present on the slide to
have the requisite data for scoring. This requires the CTC sample to have high recovery from the initial blood
sample. Once the CTCs are isolated, the transfer of cells to the pathology slide must be very efficient since there
are far fewer cells present as compared to tissue slices. It is advantageous if the CTCs are concentrated to a
relatively small area on the slide to minimize scanning time. The analysis hardware and software need to maintain
exceptional resolution and image processing capabilities to elucidate data from CTC samples.
IsoFlux System for CTC Analysis
The IsoFlux System provides access to CTCs from whole blood and other biological samples. The platform utilizes
a proprietary microfluidic device and immunomagnetic particles to isolate intact CTCs and delivers them with high
recovery, high purity, and low elution volume (<20µL). IsoFlux CTC samples can be readily transferred to pathology
slides for FISH analysis. The samples are deposited on a 1cm2 of area of the slide which greatly facilitates both the
speed and efficiency of analysis.
GenASIs Scan and Analysis
GenASIs Scan & Analysis is an automated solution for detecting, classifying and enumerating signals in FISH
samples. The platform supports a plurality of probes, staining procedures and sample types. The platform includes
a robotic scanner with 9-slide or 81-slide automated slide feeder for true “walk-away” functionality. This capability
allows for the automation of slide analysis, documentation and reporting. The system supports manual, semiautomated and fully-automated flows to suit any labs needs and budget. The GenASIs suite of platforms addresses
the needs of modern FISH laboratories by offering solutions to the challenges laboratories often face when
processing FISH slides.
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Cytogenetic Monitoring of Cancer Progression in Circulating Tumor Cells
Utilizing the IsoFlux System and GenASIs Spectral Imaging Technology
APPLICATION NOTE
Methods
Sample collection and preparation
Peripheral blood samples from breast cancer patients were drawn (8-10mL, EDTA) and shipped fresh overnight for
CTC isolation. Temperature controlled shipping packs were used to maintain sample integrity. Sample was
fractionated using density centrifugation (Ficoll) to isolate the white blood cell fraction. These cells were resuspended in 500µL of buffer and coupled with IsoFlux immunomagnetic particles at 4˚C under rotation.
Two representative cells identified in
a clinical CTC isolate from a breast
cancer patient. Cell (top) showing a
normal Her2 chromosomal pattern
(green probe). Cell (bottom) with
amplification of Her2.
IsoFlux System for CTC Access
Prepared samples were dispensed into the IsoFlux microfluidic cartridge and loaded into the IsoFlux benchtop
instrument. The sample isolation protocol was implemented to separate CTCs from the background cell population.
The cartridge was designed to use a flow-through channel and an optimized magnetic isolation zone for maximum
efficiency and purity. An online wash step is integrated into the standard protocol. The sample was automatically
transferred to a microcentrifuge tube in less than 20µL. The entire sample was placed on a poly-l-lysine coated
glass slide within1cm2 circle demarcated using a hydrophobic marker. The sample was air dried and fixed using 3:1
methanol:acetic acid solution prior to hybridization.
FISH Probe Application
The prepared slides were dehydrated in Ethanol series (70%; 85% and 100%). Slides were left to dry and then
hybridized with the Vysis TOP2A/HER-2/CEP 17 probe
kit (Red, Green and Aqua respectively) according to the
manufacturer protocol (Abbott Laboratories, Illinois,
U.S.A). After 16h the slides were washed and then
stained with DAPI (4',6-diamidino-2-phenylindole).
FISH Imaging and Scoring
The sample was scanned using GenASIs Scan and
Analysis system (Applied Spectral Imaging). CTC
location was determined using an automated BX61
microscope with a 40x objective, a 9-slide motorized
Marzhauser stage, and DAPI filter cube. The sample is
A until the required number of cells is reached.
scanned
Dedicated filters that match the probe were used to
capture the signals in multiple focal planes. Cells were
classified based on their signal pattern which identifies
clinical status.
About Applied Spectral Imaging
Applied Spectral Imaging (ASI) makes patient care better through advanced biomedical imaging. With
superior imaging and analysis capabilities, ASI provides state of the art diagnostic aids, offering cytogeneticists and pathologists accurate analysis. GenASIs enables automated tissue analysis for primary diagnostics,
with reproducible and reliable results. GenASIs is FDA cleared for FISH clinical applications such as
UroVysion, HER2/neu , CEP XY and Karyotyping. ASI is the industry's leading microscopy imaging solution
provider since 1993 with over 2,500 systems deployed worldwide.
5315 Avenida Encinas, Suite 150
Carlsbad, CA 92008
T: 760.929.2840
TOLL FREE: 800.611.3466
www.spectral-imaging.com
About Fluxion Biosciences
Fluxion Biosciences provides cellular analysis tools for use in critical life science, drug discovery, and
diagnostic applications. Fluxion’s proprietary microfluidic platform enables precise functional analysis of
individual cells in a multiplexed format. Products include the BioFlux™ System for studying cellular interactions, the IonFlux™ System for high throughput patch clamp measurements, and the IsoFlux™ System for rare
cell analysis. Fluxion’s systems are designed to replace laborious and difficult assays by providing intuitive,
easy-to-use instruments for cell-based analysis.
385 Oyster Point Blvd., #3
South San Francisco, CA 94080
T: 650.241.4777
TOLL FREE: 866.266.8380
www.fluxionbio.com
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