be automatically stained with antibodies for identification or can be released out of the cassette for subsequent analysis. these released / harvested cells are alive and can be analyzed by downstream cellular and molecular techniques, as well as cultured. the filtration cassette captures a plethora of different cancer cell types. in may 2022, the parsortix® pc1 system was cleared by the fda as a medical device for the capture and harvest of circulating tumor cells (ctcs) from metastatic breast cancer patient blood for subsequent analysis. in addition to the ivd application, the pc1 may be used with the mbc-01 metastatic breast cancer kit for use in research studies or lab developed tests (ldts) that have been created and validated in a clinical laboratory. epic sciences method this method involves technology to separate nucleated cells from red blood cells, which lack a nucleus. all nucleated cells, including normal white blood cells and ctcs, are exposed to fluorescent-tagged antibodies specific for cancer biomarkers. in addition, epic's imaging system captures pictures of all the cells on the slide (approximately 3 million), records the precise coordinates of each cell, and analyzes each cell for 90 different parameters, including the fluorescence intensity of the four fluorescent markers and 86 different morphological parameters. epic can also use fish and other staining techniques to look for abnormalities such as duplications, deletions, and rearrangements. the imaging and analysis technology also allows for the coordinates of every cell on a slide to be known so that a single cell can be retrieved from the slide for analysis using next-generation sequencing. a hematopathology-trained algorithm incorporates numerous morphology measurements as well as expression from cytokeratin and cd45. the algorithm then proposes candidate ctcs that a trained reader confirms. cells of interest are analyzed for relevant phenotypic and genotypic markers, with regional white blood cells included as negative controls. epic's molecular assays measure protein expression and also interrogate genomic abnormalities in ctcs for more than 20 different cancer types. maintrac maintrac is a diagnostic blood test platform applying microscopic in vitro diagnostic methods to identify rare cells in body fluids and their molecular characteristics. it is based on positive selection using epcam-specific antibodies. maintrac uses an approach based on microscopic identification of circulating tumor cells. to prevent damage and loss of the cells during the process, maintrac uses just two steps towards the identification. in contrast to many other methods, maintrac does not purify the cells or enrich them, but identifies them within the context of the other blood compounds. to obtain vital cells and to reduce stress of those cells, blood cells are prepared by only one centrifugation step and erythrocyte lysis. like cellsearch, maintrac uses an epcam antibody. it is, however, not used for enrichment but rather as a fluorescent marker to identify those cells. together with the nuclear staining with propidium iodide the maintrac method can distinguish between dead and living cells. only vital, propidium excluding epcam positive cells are counted as potential tumor cells. only living cells can grow into tumors, therefore dying epcam positive cells can do no harm. the suspension is analysed by fluorescence microscopy, which automatically counts the events. simultaneous event galleries are recorded to verify whether the software found a true living cell and to differentiate between skin epithelial cells for example. close validation of the method showed that additional antibodies of cytokeratins or cd45 did not have any advantage. unlike other methods maintrac does not use the single cell count as a prognostic marker, rather maintrac utilizes the dynamics of the cell count. rising tumor cell numbers are an important factor that tumor activity is ongoing. decreasing cell counts are a sign for a successful therapy. therefore, maintrac can be used to verify the success of a chemotherapy and to supervise the treatment during hormone or maintenance therapy maintrac has been used experimentally to monitor cancer recurrence. studies using maintrac have shown that epcam positive cells can be found in the blood in patient without cancer. inflammatory conditions like crohn's disease also show increased levels of epcam-positive cells. patients with severe skin burns can also carry epcam positive cells in the blood. therefore, the use of epcam-positive cells as a tool for early diagnosis is not optimal. physical methods physical methods are often filter-based, enabling the capture of ctcs by size rather than by specific epitopes. screencell is a filtration based device that allows sensitive and specific isolation of ctcs from human whole blood in a few minutes. peripheral blood is drawn and processed within 4 hours with a screencell isolation device to capture ctcs. the captured cells are ready for cell culture or for direct characterization using viewrna in situ hybridization assay. the parsortix method separates ctcs based on their size and deformability. hybrid methods hybrid methods combine physical separation (by gradients, magnetic fields, etc.) with antibody-mediated cell retrieval. an example of this is a sensitive double gradient centrifugation and magnetic cell sorting detection and enumeration method which has been used to detect circulating epithelial cancer cells in breast cancer patients by negative selection. the principle of negative selection is based on the retrieval of all blood cells by using a panel of antibodies as well as traditional gradient centrifugation with ficoll. a similar method known as iset test has been employed to detect circulating prostate cancer cells and another technique known as rosettestep has been used to isolate ctcs from small-cell lung cancer patients. similarly, researchers at massachusetts general hospital have developed a negative selection method which employs inertial focusing on a microfluidic device. the technique, called ctc-ichip, first removes cells too small to be ctcs, such as red blood cells, and then uses magnetic particles to remove white blood cells. ctc characterization some drugs are particularly effective against cancers which fit certain requirements. for example, herceptin is very effective in patients who are her2 positive, but much less effective in patients who are her2 negative. once the primary tumor is removed, biopsy of the current state of the cancer through traditional tissue typing is not possible anymore. often tissue sections of the primary tumor, removed years prior, are used to do the typing. further characterization of ctc may help determining the current tumor phenotype. fish assays have been performed on ctc as well as determination of igf-1r, her2, bcl-2, erg, pten, ar status using immunofluorescence. single cell level qpcr can also be performed with the ctcs isolated from blood. the organ tropism of patient-derived ctc has been investigated in a mouse model. ctcs isolated from breast cancer patients and expanded in vitro showed they could generate bone, lung, ovary and brain metastases in mice, partially reflecting the secondary lesions as found in the corresponding patients. remarkably, one ctc line—isolated long before the appearance of brain metastasis in patient—was highly competent to generate brain metastasis in mice. this was the first predictive case for brain metastasis and a proof of concept that intrinsic molecular features of metastatic precursors amongst ctcs could provide novel insights into the mechanisms of metastasis. cell morphology morphological appearance is judged by human operators and is therefore subject to large inter operator variation. several ctc enumeration methods exist which use morphological appearance to identify ctc, which may also apply different morphological criteria. a recent study in prostate cancer showed that many different morphological definitions of circulating tumor cells have similar prognostic value, even though the absolute number of cells found in patients and normal donors varied by more than a decade between different morphological definitions. history ctcs were observed for the first time in 1869 in the blood of a man with metastatic cancer by thomas ashworth, who postulated that "cells identical with those of the cancer itself being seen in the blood may tend to throw some light upon the mode of origin of multiple tumours existing in the same person". a thorough comparison of the morphology of the circulating cells to tumor cells from different lesions led ashworth to conclude that "one thing is certain, that if they came from an existing cancer structure, they must have passed through the greater part of the circulatory system to have arrived at the internal saphena vein of the sound leg". the importance of ctcs in modern cancer research began in the mid 1990s with the demonstration that ctcs exist early on in the course of the disease. those results were made possible by exquisitely sensitive magnetic separation technology employing ferrofluids (colloidal magnetic nanoparticles) and high gradient magnetic separators invented by paul liberti and motivated by theoretical calculations by liberti and leon terstappen that indicated very small tumors shedding cells at less than 1.0% per day should result in detectable cells in blood. a variety of other technologies have been applied to ctc enumeration and identification since that time. modern cancer research has demonstrated that ctcs derive from clones in the primary tumor, validating ashworth's remarks. the significant efforts put into understanding the ctcs biological properties have demonstrated the critical role circulating tumor cells play in the metastatic spread of carcinoma. furthermore, highly sensitive, single-cell analysis demonstrated a high level of heterogeneity seen at the single cell level for both protein expression and protein localization and the ctcs reflected both the primary biopsy and the changes seen in the metastatic sites.