x-posted at Erica Pratt’s Blog
CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Cell Fixation: Chemical preservation of a cell. Post-fixation, a cell is no longer alive.
Cell Staining: Using different markers to visualize cells, or components of cells.
Fluorophore: A fluorescent molecule that is excited at one wavelength of light (excitation), and emits light at another wavelength (emission).
Leukocyte: White blood cell (WBC).
Pathology Slide: A fixed section of unhealthy tissue that can be analyzed with various cell visualization markers.
Phenotype: Observable characteristics of a cell.
RBC: Red blood cell or erythrocyte
Systemic disease: Disease that has spread throughout the body.
Why use microscopic characterization to identify CTCs?
CTCs, which are shed from tumors into the vasculature, are considered to be key players in metastasis, and ultimately cancer patient death. Therefore, the goal of many CTC isolation systems is to separate these abnormal (cancer) cells from normal (blood) cells. Post-capture, analyses can be performed to analyze morphological differences between individual CTCs. Additionally, more researchers are investigating an underpinning assumption in CTC isolation: are the blood cells of a patient with systemic disease normal? One method used to answer that question is high-resolution, microscopic characterization of blood samples.
This technique is unique when compared to size– or immunocapture– based sorting I described previously. Microscopic identification of CTCs does not rely on physically separating them from native blood cells; instead, it uses imaging in combination with rapid scanning to look at almost all cells present in a blood sample. Many devices use microscopy to identify CTCs post-capture, but view other blood cells as contaminants to be identified so as not to confound results. Microscopic characterization aims to look at CTCs and other blood cells to further understand the pathology of the disease1-6. This method requires extensive image processing, and cell categorization algorithms.
What platforms are used for microscopic CTC characterization?
Most techniques focused on blood cell and CTC characterization have an initial stage to remove RBCs and small WBCs, either through lysis or filtration. The remaining cells are fixed (killed), and analyzed for a number of distinguishing characteristics. I will divide techniques by the substrate used, either non-porous surfaces (i.e. glass slides), or polymer porous surfaces (i.e. microfilters).
Non-porous Surfaces, such as modified glass slides1,2,3, are used to deposit and fix blood samples after minimal pre-processing (usually RBC lysis). Multiple slides can be produced from one 10mL blood sample3, enabling researchers and clinicians to perform multiple assays on one blood draw with slides left over for storage. The example to the left is an x-y plane image of stained pancreatic CTCs and a 3-D reconstruction using multiple x-y plane images at different depths in the sample (z).
Porous Surfaces use devices like microfilters4,5,6 to eliminate RBCs and small leukocytes prior to cell fixation and analysis. Cells are captured at regular intervals along the filter, making it simple to create a registry system to store unique information about each isolated cell. Multiple filters are used for one blood draw5, enabling the same analyses and storage as in the non-porous surface case.
Both of these techniques allow for morphological analysis of different types of captured cells, while also producing images very similar to standard pathology slides, making them attractive for clinical implementation.
Continue reading “How to Sort Circulating Tumor Cells Part III: Microscopic Characterization”