A rare-cell detector for cancer

Abstract

Although a reliable method for detection of cancer cells in blood would be an important tool for diagnosis and monitoring of solid tumors in early stages, current technologies cannot reliably detect the extremely low concentrations of these rare cells. The preferred method of detection, automated digital microscopy (ADM), is too slow to scan the large substrate areas. Here we report an approach that uses fiber-optic array scanning technology (FAST), which applies laser-printing techniques to the rare-cell detection problem. With FAST cytometry, laser-printing optics are used to excite 300,000 cells per sec, and emission is collected in an extremely wide field of view, enabling a 500-fold speed-up over ADM with comparable sensitivity and superior specificity. The combination of FAST enrichment and ADM imaging has the performance required for reliable detection of early-stage cancer in blood.O ccult tumor cells (OTCs) shed from tumors can travel through the blood stream to anatomically distant sites and form metastatic disease, the major cause of cancer-related death in patients with solid tumors. These disseminated cells are present in circulation in extremely low concentrations, estimated to be in the range of one tumor cell in the background of 10 6 -10 7 normal blood cells and are occult to routine imaging and laboratory studies (1). Automated digital microscopy (ADM) using image analysis for recognition of specifically labeled tumor cells has been demonstrated to be the most reliable method currently available for OTC detection (2-5).However, at the typical scan rate of 800 cells per sec, ADM is too slow to screen for a statistically valid number of OTCs (6). This slow scan rate is a result of two factors. One is the substantial latency associated with stepping the sample under the microscopy objective. This stepping results from the lens' small field of view. The other factor is the long exposure time that is due to the low level of excitation from broadband illumination sources and the lack of sensitivity of the charge-coupled device detector used for imaging.Here we report a scanning instrument using fiber-optic array scanning technology (FAST) that can locate OTCs at a rate that is 500 times faster than ADM, with comparable sensitivity and improved specificity. The exposure time is reduced by using a laser source for higher illumination levels and a more sensitive photomultiplier detector. However, our key innovation is providing an optical system with an exceptionally large field of view (50 mm) without a loss of collection efficiency. By collecting the fluorescence in an array of optical fibers that forms a wide collection aperture, the FAST cytometer has a 100-fold increase in field of view over ADM. Although this increase in field of view comes with a reduction in instrument resolution, the resolution is still sufficient for the identification of fluorescently labeled cells. This field of view is large enough to eliminate the need to step the sample under the collection optics, and hence there is no s...

Circulating tumor cell technologies

“…However, the emergence of new data highlighting the importance of (Das et al, 2012;Krivacic et al, 2004;Somlo et al, 2011) CytoTrack CK, CD45, DAPI Special glass disc scanned as it spins, clusters observed, 100 M cells/min (Hillig et al, 2015) In vivo Photoacoustic flow cytometry (PAFC)…”

Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR

“…Immuno-PCR was performed in addition to the use of pan anti-cytokeratin (CK) and anti-CD45 primary antibodies to visualize circulating tumor cells and leukocytes, respectively. Slides were sealed with coverglass and imaged using a customized high-throughput fluorescence microscope with 10× magnification (32)(33)(34). Cells were identified using Hoechst 33342 nuclear staining and the fluorescence intensity of SK-BR-3 and MDA-MB-231 was determined.…”

“…Cells were washed two times with PBST (0.05% Tween 20) and then the antibodyoligonucleotide solution was added to the cells for 40-60 min at 37°C. (32). In addition to automated identification, WBCs spiked with both SK-BR-3 and MDA-MB-231 (labeled with CellTracker) cells were also imaged with a confocal microscope (Zeiss 710).…”

A transfer function approach for predicting rare cell capture microdevice performance

“…Finally, circulating tumor cells (CTCs), which are shed from a primary tumor into the circulatory system and are thought to contribute to metastasis (Maheswaran and Haber 2010), can inform patient prognosis (Cristofanilli et al 2004), therapeutic efficacy Stott et al 2010), and the genetics of disseminating cancer cells (Russnes et al 2010;Navin et al 2011;Pratt et al 2014;Lohr et al 2014). A challenge in studying these cells is that they are exceedingly rare-as few as 1 CTC per 100 million blood cells, for example (Racila et al 1998;Krivacic et al 2004)-requiring engineered solutions to isolate as many of the target cells as possible, while capturing few blood cells.…”