CTC enumeration

VyCAP’s CTC enumeration solution is easy, requires limited hands-on time and results in high recoveries. This size based filtration uses the larger size and rigidity of circulating tumor cells compared to other blood cells.

The CTC enumeration technology consists of :

  1. A filtration disposable, that contains a microsieve filter with defined pores (5.0 ± 0.2µm)
  2. An imaging system, to automatically image and enumerate CTC

VyCAP offers a complete, simple, validated and automated solution for the CTC enumeration of cancer patients and xenograft models. The solution contains of hardware, software and protocols. Protocols to enumerate the cancer cells in whole blood of mouse and humans are found on our website.

Unique Selling Points CTC enumeration

  • Short (<1hr) and simple workflow to enumerate CTC from whole blood
  • High CTC recoveries >70%
  • Validated protocols for Transfix (Cytomark) and CellSave (Silicon Biosystems)
  • Perfect images without auto-fluorescence
  • Dedicated imaging system CTC enumeration

CTC enumeration with VyCAP microsieve filter chip

Short and validated workflow for CTC enumeration

CTC enumeration flow
The workflow to filter whole blood from human or mouse is validated and simple. It takes less than 1 hr to process and enumerate circulating tumor cells. We advise to use Transfix or Cellsave collection tubes and VyCAP hardware (filtration disposable, cell stainer, pump-unit and imaging system) and software(Imaging system and ACCEPT) to detect CTCs. All our staining reagents described in the protocols are commercial available. Contact us or download the protocols for more information.

Filtration disposable contains chip with precisely defined pores

CTCs are larger and more rigid compared to blood cells. The difference between the CTC and blood cells such as WBCs is small. The microsieve chip is therefore manufactured using dedicated micromachining technologies in a cleanroom. The micro technology microsieve filter chip contains 160.000 precisely defined pores (pore diameter 5 ± 0.2 µm). Precisely defined pores give reliable whole blood filtration results and prevents clogging.

CTC enumeration microsieve filter

Thin microsieve membrane results in less force on cells

The filter is made from a 1µm thick silicon nitride (SiN) membrane which is supported by a structure below the membrane. VyCAP developed the thinnest CTC filter on the market. Therefore the required pressure across the microsieve filter is much lower compared to other size based filtering systems. A low pressure results in less force and less cell damage on the captured cells.

Pump unit to filter whole blood

The microsieve filter is mounted in a plastic slide that fits in the filtration disposable. The disposable clicks onto the pump unit. Whole blood is transferred to the sample side of the disposable and a small negative pressure is applied by the pump unit. Depending on the collection tube the pressure is adjusted, see our protocols. It takes typically 1minute to filter a tube of whole blood after which the cells are labelled. CTC enumeration pump unit system to filter blood

Easy cell labbeling with the Cell Stainer

After the filtering the slide is removed from the disposable filtration unit and placed into the cell stainer. Next, the labelling reagents are applied to the cells collected on the filtration membrane (Step 1). The staining reagents stay on the filter and the cells remain submerged during incubation (Step 2). To remove excess reagents the microsieve filter is pushed onto an absorbing pad that is positioned below the microsieve filter (Step 3). This process is repeated multiple times with different reagents. The cells will remain on the sieve and will not be washed away during the staining steps.Cell stainer to label Circulating Tumor Cells

Small microsieve chip for fast imaging

Our microsieve filter chips are only 8x8mm which allows fast imaging and image processing by ACCEPT or Icy software. Besides relatively small the microsieve filter chips are atomically flat and shows no auto-fluorescence. Since the filters are flat imaging and have no auto fluorescene imaging is easily done with our imaging system.

Imaging system to enumerate CTCs on microsieve

Automatically enumerate CTC, Circulating Tumor Cells or fetal cells, with the VyCAP Imaging systemWith this dedicated system slides with microsieves can be automatically imaged and processed with the software. Up to 8 microsieves with Circulating Tumor Cells can be imaged. CTC selection software can be used to enumerate CTCs. The tool is based on a Nikon Ti-2 inverted fluorescent microscope. The imaging system can be upgraded to a Puncher system.

Imaging system is fully automated

To automatically enumerate CTC the system has two high end scanning stages. The stages are used to quickly image the slides with microsieves. The two stages are automatically controlled by software. Furthermore the system has an autofocus and changes filter cubes by itself. Up to eight slides can be loaded at once.

Imaging system has high end optical components

The inverted fluorescent microscope has a Lumencor LED excitation light source and Hamamatsu CMOS scientific camera. This enables the user to acquire images of the single cells with a 10x, 20x  40x or 60x objective. After imaging there is an automated manner to select cells on interest. These cells are presented in a gallery that is automatically generated.

System with imaging protocols for CTC

After filtration and labelling, the slide is transferred to the imaging system to acquire multi color fluorescence images. The imaging system is a fully automated and has user friendly software to acquire immune fluorescent images. It images the microsieve filter using a maximum of 6 fluorescent channels and can load 8 VyCAP slides at once. Standard protocols for CTC counting based on the DNA+, CK+ and CD45- are available and additional labels can be added ( for example PDL-1 or MUC-1). Scanning a filtration slide in 4 fluorescence colors takes around 15 minutes.

Imaging system for CTC enumeration

ACCEPT for CTC detection

The images can be processed by the open source CTC image analysis program ACCEPT. This program automatically identifies CTC on different parameters using sophisticated image analysis algorithms. After processing a list of possible circulating tumor cells are presented in a image gallery. The final selection of circulating tumor cells is done by the user.


CTC enumeration software ACCEPT

ACCEPT developed in CancerID

The software ACCEPT is developed by researchers in a EU-consortium called CancerID.The Public-Private-Partnership with currently 36 partners from 13 countries aims at the establishment of standard protocols for and clinical validation of blood-based biomarkers. It brings together experts from academic and clinical research, innovative Small-to-Medium sized Enterprises (SMEs), diagnostics companies and the pharmaceutical industry.

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