How CytoTrack works

CytoTrack technology is a new kind of scanner that is specifically developed for the challenges of scanning and analyzing rare cells, such as Circulating Tumor Cells (CTC).

The basic idea behind CytoTrack is closely related to conventional CD/DVD technology. The sample is spread on a special glass disc (CytoDisc™). The area of the disc is much larger than a conventional microscope slide and can accomodate up to 100 million cells in a monolayer. This makes it possible to scan all nucleated cells in a blood sample, and hereby eliminate the need for a pre-selection step.

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The CytoDisc™ is scanned with the CytoTrack CT11™ scanner. The scanner rotates the disc at high velocity and a laser system scans the entire surface. The acquired data is processed by CytoTrack's data management system that identifies the target cells (CTCs) and captures the exact position of each target cell on the disc. After the primary scan, each target cell is revisited for image analysis and further validation.

The scanning capacity is ultra high: 100 million cells can be scanned in just one minute!

No need for pre-selection

Conventional rare cell technologies depend on a pre-selection (enrichment) step. This pre-selection is normally performed by either magnetic separation (based on EpCAM) or by filtration based on the size of the cells (filtering technique). This pre-selection step is needed to reduce the amount of cells which would be fluorescent scanned later. Conventional fluorescent image scanning is very time consuming, and the capacity is typically less than 1000 cells/sec. The basic idea behind CytoTrack is to radically increase the scanning capacity. The scanning capacity of CytoTrack is 2 million cells/sec. That is 2000 times faster.

No bias

Because there is no need for a pre-selection step, the result is bias free. CTCs are detected independently of EpCAM and the size of the cells. The detection of CTCs is purely based on fluorescent signals.

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Procedure

The CytoTrack procedure includes preparation of the blood by conventional antibody staining procedures, followed by scanning with the CT11™ scanner. 

Blood preparation

  1. A blood sample is drawn from the patient
  2. White blood cells are isolated using gradient separation
  3. The sample is stained, via immunohistochemical process
  4. The sample is spread evenly on a CytoDisc™ and immobilized
  5. The CytoDisc™ is covered with mounting medium and the CytoCover™

Enumeration

  1. The disc is scanned within the CytoTrack instrument
  2. All scanning data are processed and results are displayed
  3. Each target cell is revisited for verification by imaging

On-disc characterization

  1. The CytoCover™ is removed and the mounting medium is washed off
  2. Selected areas on the disc are stained with antibodies or probes (FISH)
  3. The mounting medium and cover are reapplied
  4. The disc is reinserted into the scanner and the cells are re-imaged

Off-disc characterization

  1. The CytoCover™ is removed and the mounting medium is washed off
  2. The disc is reinserted into the scanner and cells are revisited
  3. The cells are loosened from the surface and lifted with the CytoPicker™
  4. The cells are placed into tubes or onto slides
  5. The tubes or slides are processed by PCR or NGS

The preparation process steps are performed as a standard laboratory working process. The scanning procedure is commonly performed in just 10-15 minutes per sample.

 

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The CytoTrack assay involves a staining procedure, followed by a smearing and drying procedure. Once these procedures are completed, the scan can be performed

 

CytoPicker™: Pick up cells from disc for further characterization

The CytoTrack CT11™ incorporates CytoPicker™, a new feature for picking up cells from the disc. After enumeration, the operator can select specific cells to be isolated. A pipette system loosens the individual cell from the surface, without any background of white blood cells, and transports it to a PCR tube or another slide. The cell can then be anayzed with single cell characterization techniques, such as PCR or sequencing.

 

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After detection and enumeration, it is possible to isolate the individual intact cells for characterization. Cells are easely picked up from the disc with a micromanipulator, and the cell can be entered into a tube or placed on a microscope slide (with grides).

 

Combination of 'flowcytometry' and scanning microscopy

CytoTrack technology combines the technical virtues of flowcytometry (high capacity scanning) and scanning microscopy (detailed image analysis). It can be viewed as an alternative type of flowcytometry where the cells—instead of being carried by a fluent buffer string—are attached to the surface of the CytoDisc™. In both traditionel flowcytometry and the CytoTrack cytometry, the cells are guided through the laser beam detection system with a high capacity. The CytoTrack solid state concept is unique in the way that it facilitates the necessity to revisit each target after scanning to verify each signal and to perform further single cell analysis. This is not possible when using tradional flowcytometry.

Eliminating magnetic separation

The most common technologies used for CTC detection combine magnetic separation (step 1) and fluorescent scanning (step 2). Magnetic separation is used as a pre-selection step that reduces the amount of cells to be fluorescent scanned. This is a necessary due to the limited scanning capacity of conventional scanning systems.

In order to perform magnetic separation, it is neccesary to utilize a surface marker on the cells. The most common surface marker used is EpCAM. But using EpCAM poses risks, since the expression of EpCAM varies between individual cancer cells and different types of cancer cells, and some cancer cells do not show any EpCAM expression at all.

CytoTrack technology eliminates the need for EpCAM pre-enrichment because it is based on a fluorescent scanning principle that has an extremely high scanning capacity. CytoTrack can scan 100 million blood cells within just one minute. CytoTrack therefore eliminates the magnetic separation step completely and provides a robust and efficient process based on direct scanning of CK (FITC) and other fluorescent signals to identify the target cells.

Large sample sizes improve the statistics

One of the major challenges of CTC detection is the highly limited number of target cells available. Most publications are based on a sample size of 7.5 ml blood and describe a threshold of 5 CTCs. Statistically this is weak and therefore inconclusive. Because of the high capacity of CytoTrack technology, it is straightforward to perform detections on larger sample sizes (15-30 ml). The larger sample sizes improve statistics and increases the validity of CTC detection which is especially important within frontier research use.

Key features

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EpCAM independent

All nucleated cells in the blood sample are scanned. Due to the extremely high scanning capacity of the CytoTrack scanner, the need for pre-selection is eliminated. No need for a pre-selection step based on EpCAM (magnectic separation) or filtration(based on cell size).

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Downstream analysis

After enumeration, selected target cells can be molecular characterized. Either 'on-disc' by IFA (fluorescent antibodies) and FISH (fluorescence in situ hybridization). Or 'off-disc' by isolating the target cells and performing single cell PCR or sequencing. 

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Single cell isolation

Individual intact cells can be picked up with the CytoPicker™. After enumeration scanning, the operator can select specific cells to pick up from the disc, and place them into PCR tubes.

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Ultra high scanning capacity

The CytoTrack detection principle has an extremely high scanning capacity. The scanning principle is comparable with flowcytometry. Cells are scanned optically for the fluorescent signal. After the first scan, each 'hotspot' on the CytoDisc™ is revisited and further analysed by imaging. The instrument automatically images each 'hotspot' and pictures are shown in an image gallery. This makes it easy for the operator to select CTCs from debris.

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No size filtration

Due to the ultra high scanning capacity, a pre-selection of cells by filtration is not necessary. The scanning principle is solely based on fluorescent signals, the bias of size is therefore eliminated. Many CTCs have the same size as white blood cells.

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HD imaging

The instrument is build of the highest quality optical components and provides HD (high definition) images of each individual cell. This makes it easy for the operator to determine the morphology of the cell and staining characteristics.

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Open platform

CytoTrack is an open platform that allows researchers to develop their own assays and protocols. Some of our clients experiment with their own combinations of antibodies, special GFP (green fluorescent protein), special PLP/PLA probes, and/or other types.

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Large sample size

Because CTCs are very rare cells, it is important to process large sample sizes in order to improve the statistics. CytoTrack can handle sample sizes of 15-30 ml of blood.