There are a number of technologies which use filters of different pore sizes to isolate cells based on their size. Please find detailed info here about these platforms

http://bit.ly/U0VYqh

I used this kit for proof-of-principle experiments to isolate CTCs. You can isolate efficiently "big" CTCs, but there might be smaller or less stiff cells (e.g. cells with a more mesenchymal-like phenotype) that might flow thorugh the poruses. Another problem is purity, which is not very high. Culturing them is another big issue, but to me the most interesting thing is that you can implant the filter subcutaneously in a mouse to generate CTC-derived tumors.

One of the best technique is the Scattering of x-rays, neutron and LASER.

Hello Jacob

Thank you for sharing your experience on ScreenCell filter.

There are two issues which I do agree with you.

First, the filter pore size is about 8 um if I am right thus anything less than 8 um in size would be pass through the filter. Only cells larger than 8 um will then be trapped on filter including white blood cells? For me if only one CTC cell could be held on filter, it would be worth fighting for.

Second is that isolation is not 100% pure in CTCs. Same here at our lab right now we are using electric field trying to isolate CTCs from patient blood the results always show that blood cells are every where in the isolated CTCs sample. However, we can transfer isolated CTCs sample to culture media and keep them in incubation for few days. By doing this way we can saparate blood cells from CTCs since blood cells won't attach on plate and then be skimmed off.

The only good thing is implantation directly on mouse skin. I received one picture from ScreenCell showing CTCs on filter growing on mouse skin directly this would remind me that isolated CTC is still alive and can be cultured.

This is all my thought, is not based on my experience on the filter method.

Need more inputs from others.

Thank you

In the case of screencell, I believe the pore sizes are 5.5 and 6.5 um for unfixed and fixed cells, respectively, not 8 um. However, a smaller pore size has the downside of catching more WBCs as well. The other downside is the unmodulated shear force on the cells during filtration, which might lead to target cell destruction.

Thank your Abdur for your pore size info.

Your issue on shear force is interesting. I never used ScreenCell so I do not know the process during filtration.

Do we need to pull or push a device to create force making blood sample passing through filger? or we just let it flow through filter?

Its a pulling force generated by a vaccutainer tube beneath the filter.

Do you need to lyse RBC before filtration?

If I remember propperly, the ScreenCell kit is provided with a RBC lysis buffer and you dilute the blood sample in this buffer before running it through the filter. So RBC lysis is one of the steps of the CTC isolation with the ScreenCell filter.

I would like to correct myself that the pore sizes are 6.5 and 7.5 um respectively for fixed and unfixed cells.

do check out lots of other interesting filtration based techniques and other label free techniques in this 2013 review

http://bit.ly/YyHvPh

I have worked with CTC isolation and detection for some time. As per my observation, the CTCs have a range of variable sizes so size based CTC isolation may not be the best solution. Please look at this paper

Asghar et. al. "Electrical fingerprinting, 3D profiling and detection of tumor cells with solid-state micropores", Lab chip 2012.

And its also important to consider that what type of CTCs are you isolating, some types might have less variations in sizes and elasticity.

But it would be interesting to implant the filter subcutaneously.

Catching CTC using a filter is not so straight forward as it may appear. Cells are not hard spheres and cells with a diameter that is larger than the pore diameter will pass through the pore as well.

Whether or not a cell will be able to pass through a pore is dependent on many factors, i.e., the length of the pore (thickness of the membrane), the used pressure, hydrophilic or hydrophobic properties of the sieve material, the flexibility of the cell, etc.

The optimal filter for the detection of CTC collects most CTC, low amount of leukocyte and no erythrocytes, using a low pressure. We use silicon microsieves with a very thin membrane (

Thank you Arjan for your nice comments.

It is interesting to know that CTCs are not hard object which can pass through tiny pore, smaller than their diameter. I haven't thought of it.

IMO, I think if we use relative low pressure like vacuum tube this would not be cause CTCs to pass through.

However, we may have lymphocytes left on filter but this is not much problem if we transfer filter to culture unit. By adding culture media, lymphocytes will float in media while CTCs should be grow on filter.

Based on your experience, what is the best method to isolate CTCs from patient blood?

As far as I have learnt from the internet there is no single method is good at CTCs isolation rather it would be enrichment at first method then next method will help to identify CTCs.

A vacuum pressure tube is not relative low pressure. I believe most vacuum tubes have a vacuum level of approximately 700 mbar! Besides this the vacuum in the vacuum tube reduces very quickly when it fills with fluid and the vacuum has reduced to zero when the flow stops.

We only use 10 - 50 mbar to filter whole blood using microsieves and our developed system keeps the pressure constant during the filtration.

Culturing is a possibility to discriminate the tumor cells from leukocytes but culturing is difficult and many CTC will not grow.

You are right that there is no single method that will collect and identify all CTC in whole blood for all different type of tumor cells. All methods, procedures and technologies have their own pros and cons and unfortunately there is currently no best method.

Arjan, could you please direct me to your publication on developed microsieves system? I am interested to learn from your works.

You are right about difficulty of CTC culture. As far as we go on trying to grow whatever cells we thought they were tumors, the results are dead cells within 1 month. You can see it picture from this topic. https://www.researchgate.net/post/What_type_of_cell_is_it_image_attached?ch=reg&cp=re72_x_p2&pli=1&login=sittisak@cri.or.th#view=50e55273e5438fd31200000c

The method which we are trying to isolate CTC is by applying electric field to sample. Cancer cells will travel in different direction from blood cells then be collected for further analysis. It is similar to a commercially available apparatus here http://www.apocell.com/ctc-technology-2/apostreamtm-technology/

Please take a look at our website www.vycap.com

We have developed a new filtration method for the isolation and fluorescence labelling of CTC.

Please take a look at our website. A demonstration of the method can be viewed here : http://vycap.com/Whole%20blood%20filtration.wmv

Hello Arjan,

Thank you very much for the VDO clip showing how VyCap works.

Some questions I want to know;

1. how much blood volume can be used to filter per kit?

2. Is EDTA blood in VDO patient blood or cell culture-spiked blood?

3. What is isolated cells capacity per slide? Let say that in 7.5 mL of tumor patient blood has 20 CTCs. Can we use total 7.5 mL blood in one kit? However, from the VDO I guess blood used was around 2 mL if we estimate 1 CTCs per mL blood then the filter capacity can hold 20 CTCs for 7.5 mL blood? or do we need to separate blood into several parts, 2 mL each part?

Apart from the isolation technique, I am also concerning about which tumor stage should we first try? IMO, M1 stage should provide us a lot more CTCs to isolate? If we select wrong tumor patient then we won't see any CTCs left on filter? Is this right?

Sittisak,

Some answers to your questions:

1. The volume is limited by the size of the waste container which in this case can hold max 40 ml.

2. In the video we used EDTA undiluted blood. The filtration method will as well work for blood drawn in CellSave tubes (Veridex). Spiking or no spiking is not of influence.

3. The volume of blood used in the video was about 15 ml. The number of CTC that can be collected on the sieve is determined by the surface of the sieve. However the surface is large enough to hold more than 20.000 CTCs without influencing the flowrate through the microsieve.

You are right that more CTCs are found in patients with M1 stage tumors. However not all patients with M1 stage tumors do have CTC (or maybe have CTC but these are at least not detected). It is not about selecting the right patient it is more about slecting the right method. Immuno-magnetic isolation of CTC will most probably result in a different CTC count compared to filtration.

Hi Arjan,

It is wonderful if we can use 15 mL of patient blood in one filtration. The best on top of this is, microsieve can hold twenty thousand CTCs in one filtration!. This would safe cost and increase opportunity to capture few CTCs.

What about doing ICC-F after isolation? I have a plan to do ICC-F for identifying whatever cells trapped on microsieve. Can we just remove microsieve and fix it on glass slide and do ICC-F?

Hello Sittisak,

The filter is already mounted in a plastic slide which fits under the microscope.

The captured cells can be stainend with fluorescence labels on the microsieve as demonstrated in the movie.There is no need to remove the sieve. It actually was designed such that capture, staining and imaging can be done on the sieve while it remains in plastic slide that is used for filtering.

Hello Arjan,

How about establishing new cell lines, can we transfer microsieve to cell culture plate for doing cell growth?

Yes you can.

In my oppinion, staining is much more effective than isolation. You wouldn't isolate red blood cells for a blood picture, too. This is why I changed research from isolation, whree I was at MD Anderson in the very beginning of research, to staining. At Circualte Bostin (former World Circuating Tumor Cell Summit) it was claimed to look for the waste because the isolation methods lose too much cells and are not specific enough.

With vital Tumorspheres growing from CTCs, we could already do sensitivity testing.

If you would have as many CTC as red blood cells I would fully agree that staining only is the preferred method without pre-enrichment. Unfortunately the CTC are rare, EpCAM positives and EpCAM negatives and pre-enrichment is required. The method of enrichment is a matter of the choice but enrichment is required. Enrichment of CTC by filtration is probably the easiest method. Although you will lose the smaller CTC this is not the limiting factor in the detection of CTC. Errors in staining and identification of the CTC are the most prominent sources of errors in the detection of CTC. Take the most easy enrichment method and focus on defining and validating a staining and identification protocol.  

Article Statistical considerations for enumeration of circulating tumor cells

I do NOT agree. Pre-enrichment, sortation and isolation is the reason why so many cells are lost and most of the CTC techniques are claimed to be inefficient. I was working with magnetic beads, too, but the results were not good enough. I do not claim that all EpCam positive cells are able to metastasize, but the clinical outome proves us right. All our results are already published and validated.

Article Comparing Sequential Steps For Detection Of Circulating Tumo...

Article Standardized quantification of circulating peripheral tumor ...

Thank you Katharina and Arjan for your discussion.

For my research, my aim is to establish a new cell line from CTCs in patient bloods.

It seems to me that only isolation with filtration could be a good technique to have tumor grown in cell culture.

Please share if anyone can grow isolated CTcs in vitro successfully.

Yes we can.

We already published at ecancer, and presented at IFCC WorldLab Istanbul 2014 and at EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutic, Barcelona, 2014

Conference Paper A SUBPOPULATION AMONG CIRCULATING EPITHELIAL TUMOR CELLS IN ...

Article Heterogeneity of circulating epithelial tumour cells from in...