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...

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A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1‐209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding β‐galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1‐209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high‐level induction. Under these conditions, β‐galactosidase specific and volumetric activities were 4.2‐ and 5.5‐fold higher, respectively, than those for the “GAL1” nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter‐based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 408–416, 2000.

We evaluated the analytical and clinical performance of a novel circulating tumor cell (CTC)-based blood test for determination of programmed death ligand 1 (PD-L1) protein expression status in real time in treatment-naïve non-small cell lung cancer (NSCLC) patients. CTCs were detected in 86% of patients with NSCLC (I–IV) at the time of diagnosis, with a 67% PD-L1 positivity rate (≥ 1 PDL + CTC). Among 33 NSCLC patients with PD-L1 results available via both tissue immunohistochemistry (IHC) and CTC assays, 78.9% were positive according to both methods. The CTC test identified an additional ten cases that were positive for PD-L1 expression but that tested negative via IHC analysis. Detection of higher PD-L1 expression on CTCs compared to that in the corresponding tissue was concordant with data obtained using other platforms in previously treated patients. The concordance in PD-L1 expression between tissue and CTCs was approximately 57%, which is higher than that reported by others. In summary, evaluation of PD-L1 protein expression status on CTCs isolated from NSCLC patients is feasible. PD-L1 expression status on CTCs can be determined serially during the disease course, thus overcoming the myriad challenges associated with tissue analysis.

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.

The authors have constructed an array of 12 piezoelectric ejectors for printing biological materials. A single-ejector footprint is 8 mm in diameter, standing 4 mm high with 2 reservoirs totaling 76 µL. These ejectors have been tested by dispensing various fluids in several environmental conditions. Reliable drop ejection can be expected in both humidity-controlled and ambient environments over extended periods of time and in hot and cold room temperatures. In a prototype system, 12 ejectors are arranged in a rack, together with an X-Y stage, to allow printing any pattern desired. Printed arrays of features are created with a biological solution containing bovine serum albumin-conjugated oligonucleotides, dye, and salty buffer. This ejector system is designed for the ultra-high-throughput generation of arrays on a variety of surfaces. These single or racked ejectors could be used as long-term storage vessels for materials such as small molecules, nucleic acids, proteins, or cell libraries, which would allow for efficient preprogrammed selection of individual clones and greatly reduce the chance of cross-contamination and loss due to transfer. A new generation of design ideas includes plastic injection-molded ejectors that are inexpensive and disposable and handheld personal pipettes for liquid transfer in the nanoliter regime. (Journal of Biomolecular Screening 2004:85-94)