Technology

CTC enumeration

VyCAP’s solution for the identification and enumeration of CTC is characterized by its ease of use, limited hands-on time and high recoveries. It is based on filtration and uses the larger size and rigidity of CTC compared to other blood cells to collect these cells. The difference of these two parameters between CTC and other blood cells is however small and it requires very precisely defined filtration pores to collect the CTC from the blood. Our filters are made from silicon which provides a well-defined filter that shows no auto-fluorescence and allows to acquire high quality fluorescence images of the collected CTC.

The technology consists of :

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

VyCAP offers a simple solution for researchers in university hospitals and research institutes for the enumeration and analysis of CTC in whole blood of cancer patients and xenograft models.

Unique Selling Points

  • Short (<1hr) and simple workflow to enumerate CTC from whole blood
  • High CTC collection efficiencies >70%
  • Validated protocols for CTC detection using Transfix (Cytomark) and CellSave (Silicon Biosystems)
  • Perfect images due to the atomically flat microsieve filter surface without auto-fluorescence
  • Dedicated imager with software for CTC enumeration

 

Typical workflow for CTC enumeration

Filtration disposable with microsieve chip

CTC are in general a bit larger and more rigid compared to blood cells. However the difference in size and rigidity between the CTC and other blood cells is small and separation based on these parameters requires a filter with very well defined pores. Our microsieve is manufactured using MEMS and micromachining technologies which results in a very well defined filter with precisely defined pores.

The microsieve filter contains 160.000 precisely defined pores, with a diameter of 5 ± 0.2 µm, in a silicon nitride (SiNi) membrane with a thickness of 1µm, in an effective area of 8×8 mm. Because of the thin membrane the flow resistance of the microsieves is very low compared to comparable size based filtering systems. This allows to use of a low pressure resulting in less force on the captured cells and less cell damage.The thin membrane is supported by a structure below the membrane to prevent breaking. The pores are present in long lanes.

To facilitate the handling of the microsieve filter, it is mounted in a plastic slide that fits in the filtration disposable. The disposable fits onto the pump unit. Whole blood is transferred to the sample side of the disposable and a small negative pressure is applied across the microsieve chip by the pump unit.

Labelling the collected cells with fluorescence labels

After the slide is removed from the disposable filtration unit, it is placed into the staining holder. Next, the labelling reagents are applied to the cells collected on the filtration membrane (Step 1). The staining reagents will stay on the filter and the cells remain submerged during incubation (Step 2). After incubation the excess reagents are easily removed by pushing the microsieve down onto an absorbing pad that is positioned below the microsieve (Step 3). This process can be repeated multiple times with different reagents. The cells will remain on the sieve and will not be washed away and lost during these multiple staining steps.

VyCAP imaging and enumeration system

After filtration and labelling, the slide is transferred to the imaging system to acquire multi color fluorescence images of the collected CTC and other leukocytes that are collected on the microsieve chip. The imaging system is a fully automated Nikon Ti2 fluorescence microscope, supplied with a high-end scanning stage that has room for 8 slides, a Hamamatsu Orca FLASH II camera, multiple fluorescence filter cubes and a Lumencor Sola SE II light source for fluorescence excitation.

The system has dedicated, simple and straightforward software to acquire fluorescence images of the collected cells. It is designed for scanning the microsieve filter using maximum 6 fluorescence imaging channels. It uses standard protocols for CTC counting based on the DNA+, CK+ and CD45-, but has the ability to add additional labels against for example PDL-1 or MUC-1. The stage has 8 VyCAP slide positions. Scanning a filtration slide in 4 fluorescence colors takes 15 minutes.

Besides scanning the VyCAP filtration slides, it has the ability to define your own image acquisition protocols and to scan standard microscope slides, 96 well plates, C1 Fluidigm cartridge or other sample formats. VyCAP can supply adaptors for your type of sample on request.

Automatic analysis of the acquired images using ACCEPT

The acquired images can be analyzed using the ACCEPT software that identifies CTC on different parameters using sophisticated image analysis algorithms. The detected CTC are presented in a image gallery to the reader who makes the final selection and identification of the CTC.

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