Detecting Circulating Tumor Cells using Flow Cytometry - Essay Example

? Detecting Circulating Tumor Cells (CTC) using Flow Cytometry Introduction The research field was on Flow Cytometry. It aimed to establish a reliable method for counting Circulating Tumor Cells (CTC) using flow Cytometry. Flow Cytometry is a method of enumerating and examining minute particles suspended in a fluid when passed through an electronic detector. The system has a disposable chip. This chip checks for cross contamination collect analyzed sample and to freely measurement. CTC is salient biomarkers for so many cancers. There are many systems for enumeration based on either EpCAM/CD326 which express tumor cell before microscope or RT-PCR. Protocols for this system can be applied onto other systems. Cultured cancer cells spiked into normal blood got enriched with MACR EpCAM microbeads then labeled with APC instead of intracellular staining of cytokeratins. EpCAM allows enumeration of intact CTC, cellular integrity maintenance and concomitant performance. Combination of fine tuned CTC and cytometric multicolor resulted into linear relationship between input and output cell count from zero to hundred of cells. Anti CD45 mAb was used to give satisfactory signal/ noise ratio by gate exclusion of white blood cells signal. There is little influence on lungs cancer cell PC-9 viability. CTC is of greater importance because it provides stratification of Anti-tumor treatment and furthering characterization. Several researchers have shown that circulating Tumor cells (CTC) in peripheral blood are significant prognostic marker for cancer (1-5). Presence of circulating tumor cells in the peripheral blood of patients has been involved in the Tumor development and metastasis advancement. Response of therapy and evaluation of disease get predicted by change in circulating tumor cells. Several methods have been used in the CTC-enrichment and discovery, but the standard method is the FDA-approved cell search system (Veridex) (Takao, M., Takeda, K., 2011). This employs a 7.5ml of blood and involves epithelial cell adhesion molecules (EpCAM /CD360) (8)-conjugated immuno-magnetic enrichment preceded by cell imaging process using positive immuno-staining of cytokenins. Later negative immunostaining of leucocyte common antigen (CD45) and DNA staining with DAPI. The overall advantage of this method is the rapid read out of routine measurements. This is due to the fact that sizeable information gets included in the data and its capability of multicolor analysis. This method also offers precise detection limit of pure cells of approximately (10^-5). Related research Benjamin and Steven conducted research on flow Cytometry. They inferred that there has been progress in immuno-magnetic and flow cytometry. Benjamin and Steven concluded that flow cytometry and immunomagnetic can detect and characterize circulating tumor cells. They infer that flow cytometry has demonstrated prognostic importance in prostate and breast cancer. In Benjamin’s and Steven article about “ circulating tumor cells in colorectal cancer …” there are reviews regarding the historical and development information about identification and enumeration of circulating tumor cells in colorectal cancer. The presence of circulating tumor cells in patients having metastatic carcinomas get linked with poor survival predictions (Tych, Frederik, Sjoerd, Joost, Jan &Leon, 2011). According to their article based on research, image cytometer, cell tracks got developed to advance the enumeration of rare circulating tumor cells. Cell search system got used to enumerate circulating tumor cells in seven point five milliliters (7.5 Ml) of bold of nine healthy controls and sixty eight patients. The results were obtained from cell search system were analyzed again using image cytometer. Then automated categorization of events was executed by random forest process using Matlab. An automatic classifier got developed to categorize events into apoptotic CTC, leukocytes, CTC, CTC debris and those not linked with CTC. Five reviewers agreed with categorization got from automated classifier (Gerges, N., Rak, J., Jabado, N., 2010) These two studies are linked to this article in that they both deal with circulating tumor cells. The both articles objectives are to acquire efficient and effective method of counting circulating tumor cells. Different studies show that there is a growing demand to get a reliable and comprehensive method of analyzing and enumerating CTC. Materials and methods Cell culture- Dr. Fumiaki Koizumi gifted tumor cell lines and T-lymphobladtoid cell line Molt-4. RPMI 1640 containing 10% Invitrogen was used to culture cells. Semi-confluent cells for tumor cells were picked up washed and suspended in unsoiled culture before experiments using circulating tumor cells. Kit and antibodies- tumor enrichment kit was bought. It was used for the first evaluation of CTC enumeration. Buffers- three immune reaction buffers were used. The standard immune-reaction buffer (M-buffer), T-buffer which has increases efficiency and has antifoaming effect and the F-buffer which decreases sedimentation of cells during measurements. Competitive assay There was an examination of five EpCAM mAbs, 9C4, EBA-1, 323/A3, VU-1D9 and O.N277. This was done to determine whether a PC-9 cell binding is competitive of APC-conjugated EBA-1 or APC-conjugated 9C4. Blood Collection and Red Blood Cells Lysis Under informed consent, blood was drawn from a healthy donor. The first milliliters were discarded in order to prevent contamination with cells from blood vessels and skin. CTC tests were diluted and used to blood spike. The measurement of spiked cells was conducted using hemocytometer and quantified using flow cytometer. Immuno-magnetic CTC separation and labeling When lysing red blood cells, a reaction buffer consisting of 10-11 EpCAM-APC mAb, 20-11 FcR blocking reagent in T-buffer and 20-11 EpCAM-APC mAb was prepared. The spreading of peripheral blood single nuclear cell pellet in conical tube got transferred to 1.5 ml tube. Bottom of the tube got rinsed with 0.5ml and then combination of the rise in same 1.5ml tube. The mixture got incubated for 30 minutes at 48C. MACS MS column was used for positive selection with M- buffer substituting the T-buffer. Centrifuging was also eliminated because nano size could not influence fluorogram. Live/dead Staining and CD45 Negative staining The sample cells collected using positive selection method got centrifuged at 3003g for 5 minutes. They are removed without causing disturbance to the cell pellet. Cell viability reagent got prepared while centrifuging. Examination of CTC viability is done by DNA-binding dyes. At room temperature cells are mixed with 1ml of anti CD45APC-CymAb for 5 minutes. Setting of flow cytometer FISHMAN-R flow cytometer has blue and red laser and four detection channels. FISHMAN-R uses a disposable micro fluidic chip. The chip recognizes measurement that is precisely contamination free, sample collectability and comprehensive volume measurement. Microfluidic chip is in a holder of autoclave cassette then inserted flow cytometer. Liquid flow in the chip is generated by air pressure. Counting of signals was done at peak mode. Results and comparison to other research conducted. Flow cytometer FISHMAN-R was used to enumerate CTC within the blood. FISHMAN-R has got a disposable micro-fluidic chip for the measurements. Microfluidic chip contains all fluidic and optical paths in a closed system intended to realize cross contamination, free measurements and redirect the measured sample to further downstream experiments. Sample cells travel with the shear near the center of the chip and enter the output reservoir separated from wasted sheath. Cell search system uses EpCAM-immunomagnetic enrichment to capture CTC. The ferrofluid particles, MACS EpCAM-MicroBeads were best suited for FCM because of the size of the beads of (10-20µm). Light scattering due to these particles can be controlled beneath the detectable level with loss of information concerning blood cells and CTC. There have been reports concerning success enrichment of circulating tumor cell (CTC) from the PBMC of human blood using the EpCAM-MicroBeads (21-24). EpCAM-MicroBeads were investigated as a suitable method for CTC enrichment cell search system which utilizes intracellular staining with fluorescent ant-ck8, 18 and 19 mAbs after cell fixation. Circulating Tumor cells (CTC) in peripheral blood of patients has led to progression and metastasis development (Takao, M., Takeda, K., 2011). This has been hinted in various research works. Thomas 1869 hinted that cells similar to those of cancer tend to shed light ahead the mode of origin of multiple tumors. This is seconded by a research from the book Detection of circulating tumor cells in peripheral blood with automated scanning microscope (Ntouroupi, 2008) where he concurs with Thomas. This research findings show that there is light scattering to particles. It also found out that light scattering can be controlled beneath detectable level. Unlike these two researches which uses automatic scanning microscope, this research focused on FISHMAN-R Flow cytometer.  The research concluded that EpCAM-MicroBeads were a suitable method for CTC enrichment cell search system which utilizes intracellular staining with fluorescent ant-ck8, 18 and 19 mAbs after cell fixation. It is evident that CTC in peripheral blood of a patient normally leads to tumor progression and development of metastasis (Pantel, Alix-Panabieres & Riethdorf, 2009). Pantel, Alix-Panabieres & Riethdorf research focuses on tumor progression and development of metastasis. This research is focused on enumeration of CTC using the flow cytometry. The similarity of this research with these other researches is that they deal with better and effective detection procedures. Benjamin and Steven conclusion that flow cytometry and immunomagnetic can detect and characterize circulating tumor cells is similar to this research finding. Tych, Frederik, Sjoerd, Joost, Jan &Leon, 2011 research, image cytometer, cell tracks got developed to advance the enumeration of rare circulating tumor cells. Cell search system got used to enumerate circulating tumor cells. There research enumerated circulating tumor cells using image cytometry unlike this research which used flow cytometry.s Critical appraisal Fundamental principles of flow cytometry have been emphasized. Measurements and CTC analysis have been extensively summarized. Difficulty of cell preparation techniques and results interpretation has been discussed comprehensively in this article. It also infers that some tumors and organs might be of prognostic value. There are some fundamental issues not discussed such as other tumor makers usefulness, aneuploid and diploid tumors. Conclusion A simple protocol of concomitant enumeration and live/dead validation of CTC using FCM was developed. It was noticed that technologies of cell search all rely on the epithelial marker EpCAM for cell enrichment or detection. Ferrofluid particle is biologically inert and does not interfere with FCM measurements. EpCAM double-positive selection method designed to detect intact CTC FISHMAN-R FCM using a microfluidic chip features across contamination-free measurements, enumeration oof the CTC and permits collection of the measured sample. Antibody used for cell labeling (EpCAM-APC) appeared to be partially competitive with the EpCAM –MicroBead-ads and so it should be improved. The process would be better if it was fully automated. References Takao, M., Takeda, K., 2011. Cytometry. Enumeration, Characterization, and Collection of Intact Circulating Tumor Cells by Cross Contamination-Free Flow Cytometry, p.107-117. Gerges, N., Rak, J., Jabado, N., 2010. New technologies for the detection of circulating tumor cells, p.49–64. Pantel K., Alix-Panabieres C., Riethdorf 2009. Cancer micro metastases. Mumbai: India Negin, B. P., Cohen, S.J., 2010. Gastrointestinal Malignancies. Circulating Tumor Cells in Colorectal Cancer: Past, Present, and Future Challenges, p. 1–13. Scholtens, T. M., Schreuder, F., Sjoerd T., 2012. Cytometry. Automated Identification of Circulating Tumor Cells by Image Cytometry, p.138-148. Wang L, Wang Y, Cheng M, 2009. Flow cytometric analysis of CK 19 expression in the peripheral blood of breast carcinoma patients, China. Ormerod, M. G., 2000. Flow cytometry: A practical approach. Oxford: Oxford University Press Ntouropi, T.G. ed., 2008. Detection of tumor cells in peripheral blood with an automated scanning fluorescence microscope, Net library database. Armstrong, A. J., 2011. Molecular cancer research. Circulating Tumor Cells from Patients with Advanced Prostate and Breast Cancer Display Both Epithelial and Mesenchymal Markers, p. 580-586. Paterlini-Brechot P, Benali, N.L., 2007. Circulating tumor cells (CTC) detection. Clinical impact and future directions, p.180–204. Read More