Wednesday, May 22, 2013

Chemosensitivity testing using CTCs from patient samples

Chemosensitivity Testing of Circulating Epithelial Tumor  Cells (CETC) in Vitro: Correlation to in Vivo Sensitivity  and Clinical Outcome
www.laborpachmann.de/pdf/Chemosensitivity_JCT_2013.pdf

interesting paper and opens up new application area for CTCs.

Method for isolation: RBC lysis followed by image analysis of all nucleated cells

Conclusion: "Thus, we are able to show that chemosensitivity testing of circulating tumor cells provides real-time information about the sensitivity of the tumor present in the patient, even at different times during therapy, and correlates with treatment success. "

Thursday, May 2, 2013

PLOS publication comparing the performance of various types of filters for CTC recovery


Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood


This is great! I have been wanting to do this myself for quite sometime. There are tons of CTC technologies based on using a filter of one type of another to recover CTCs. In fact this probably is the most crowded sub-segment of commercial CTC platforms. This is mainly due to low technical threshold needed to get to market. Thus attracting lots of market entrants.

However, not many have attempted a broader study of exactly what the correlation is between the porosity, shape and size of pores to the precisely measured cellular dimensions from cell lines as well as patient samples. This study has attempted to do it. Thanks to the authors.

Key findings:

Cell Enumeration is Easiest on a Stiff Filter with Low Porosity

>> The authors point out that there is difficulty in imaging a flexible substrate, which is intuitive, hence rigid substrates are preferred.

>> "The high porosity and low number of pores of the microsieves and the TEM grids resulted in a cell density too high to reliably distinguish adjacent cells when imaged with a 4x/NA0.13 objective" ==> this is a bit confusing. my understanding is that high porosity = high number of pores per cm2. the paper seems to suggest otherwise. Share your thoughts

>> "he ideal filter should maintain its planar form during filtration, it should not react with the sample and pores should be sufficiently separated to facilitate discrimination of cells"

Increasing Pore Size Leads to Lower Recovery and Higher Sample Purity
>> This is also intuitive as larger pores deplete WBCs better than smaller pores but also lose more CTCs

>> recoveries vary for different cell lines at different pore sizes, indicating that the filter method is not one size fits all, which also intuitive, given the vast heterogeneity among tumor cells

>> "As long as there are sufficient pores to pass the sample, recovery seems to be insensitive to the number of pores. There is almost no difference between the microsieves with 115,000 pores and the one with 26,000 pores in terms of spiked cell recovery or pressure across the filter, but the number of leukocytes retained is 3.1 fold higher on the filter with 4.4 fold more pores"

Recovery is Constant Until Approximately 2% of Pores are Occupied

The Volume that can be Filtered is Limited by the Contaminant Concentration
>> that is smaller volumes lead to better recovery rates than larger volumes of sample

Monocytes are Retained More than Other Leukocytes

EpCAM+CK+CD45− CTC (from patient samples) are Smaller than Typical Cells Derived from Tumor Cell Lines

Cell Lines with Size of CTC Typically have Low Recovery

Summary:
This paper is a highly recommended read as it clarifies several aspects of membrane-based CTC filtration devices. The key points are, a) one size does not fit all, b) there is always a recovery purity trade-off in this method, and c) CTC recovery using this method is subject to a number of factors such as geometry, number of pores, rigidity, flow rate, sample volume etc.

The authors summarize it in the manuscript "In summary, the ideal filter for CTC enrichment from 10 ml of whole blood has a pore size of about 5 µm, thickness of at least 10 µm, at least 100,000 regularly spaced pores, a porosity of 10% or less and is constructed of a stiff, flat material, which does not interact with blood cells. While cell size is an important factor in determining recovery, other factors must be involved in determining whether a cell can pass as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system, such as Colo-320, SW-480 and not cell lines significantly larger than CTC."


Limitations:
though the authors used a polycarbonate track etched membrane, they did not use a patterned parylene membrane or other deformable membrane, so arrive at the conclusion that a hard surface is necessary may require additional experimentaiton  comparing the track etched polycarbonate versus parylene or other patterned substrate (this study may already exist, I will have to look it up)

the spiked cells used in the experiments were pre-stained. this is not the case for patient samples. typically, if staining were to be done on filter post recovery, a certain amount of cell loss can be expected due to this process

I wrote another post some time ago
The Problem with using size and deformability as the criteria to isolate Circulating tumor cells (CTC)

I found another good reference below
Pore design and engineering for filters and membranes