Molecular analysis of cytology specimens

Abstract

The invention features a microfluidic device including an inlet in fluid communication with a channel including an array including one or more rows of posts, wherein each row of posts includes one or more capture areas and one or more passing areas, wherein the one or more capture areas includes the posts and the passing area does not include any posts.

Description

[0001] MOLECULAR ANALYSIS OF CYTOLOGY SPECIMENS

[0002] Statement as to Federally Funded Research

[0003] This invention was made with government support from the National Institute of Health (NIH) grants R01 GM138778, R33CA281794, and T32CA079443. The government has certain rights in the invention.

[0004] Background of the Invention

[0005] The invention relates to materials and methods useful for diagnosing cancer; in particular, from paucicellular samples.

[0006] Identification of cancer in biological samples is challenging. For example, cholangiocarcinoma (CCA) is a malignant tumor arising from bile ducts. CCA is one of the most aggressive cancers, with a 5- year survival rate remaining at only 10%. Similar to a majority of cancers, clinical challenges for identifying CCA include the absence of effective screening tools, the gradual progression until symptoms manifest, and the low diagnostic accuracy of CCA tissue due to the paucicellular nature of specimens obtained during an intervention. Cholangiocarcinoma grows within and along bile ducts, ultimately causing obstruction and hepatic mass lesions in later stages of the disease. While those lesions are easily targeted for biopsy with good yields, earlier forms of malignant degenerations are much more difficult to detect, particularly in underlying sclerosing biliary disease. In these cases, endoscopic retrograde cholangiopancreatography (ERCP) is performed, and brush, balloon, or bile samples are sent for cytopathology. Unfortunately, the cellular material in these samples is often scant. Prior studies have found that up to 20% of brush biopsies are inconclusive, while the overall diagnostic accuracy hovers around 80%. These factors often lead to reexamination, lengthy genomic testing (snapshot), and delays in the treatment. Thus, improving the diagnostic accuracy of paucicellular biliary specimens as well as for other cancers is a critical step in improving treatment.

[0007] Summary of the Invention

[0008] The present disclosure relates to devices, methods and systems for the diagnosis of target cells in cytology specimens that are difficult to analyze with conventional immunohistochemistry methods, such as flow cytometry. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0009] In one aspect, the disclosure features a microfluidic device including an inlet in fluid communication with a channel including an array including one or more rows of posts. The microfluidic device may have one or more capture areas and one or more passing areas, wherein the one or more capture areas may have the posts and the passing area may not have any posts. In some embodiments the microfluidic device may have at least two rows of posts, wherein a first row of posts is adjacent to a second row of posts, and wherein:

[0010] (a) the geometric center of a first capture area in the first row does not align with the geometric center of any capture area in the second row along a direction substantially perpendicular to the rows; and / or (b) the geometric center of a first passing area in the first row does not align with the geometric center of any passing area in the second row along a direction substantially perpendicular to the rows.

[0011] In some embodiments, each row may have 1 to 50 capture areas and 1 to 50 passing areas. In some embodiments, each capture area may have two to ten posts. In some embodiments, the microfluidic device may have fifty to five hundred rows. In some embodiments, the rows of the microfluidic device are substantially parallel. In some embodiments, the rows of the microfluidic device are such that

[0012] (a) each passing area is larger in size in a direction parallel to the row including the passing area than the distance between any two adjacent posts in a single capture area;

[0013] (b) the size of a passing area in a direction parallel to the row including the passing area of a first row of posts is different from the size of a passing area in a direction parallel to the row inculding the passing area of a second row of posts; and / or

[0014] (c) the distance between any two adjacent posts in a single capture area of a first row of posts is different from the distance between any two adjacent posts in a single capture area of a second row of posts.

[0015] In some embodiments, the size of a passing area is in a direction substantially parallel to the row including the passing area is a first distance and the distance between any two adjacent posts in a single capture area is a second distance.

[0016] In another aspect, the disclosure features a device with an inlet and a channel in fluidic communication with the inlet, wherein the channel may have an array of posts positioned in one or more rows with a first distance and a second distance between adjacent posts in each row. In some embodiments of the device, the first distance is at least 20 pm. In some embodiments, the first distance is about 45 pm. In some embodiments, the second distance is from about 15 pm to about 25 pm. In some embodiments, the second distance is about 22.5 pm. In some embodiments, each post is T-shaped, L- shaped, triangular shaped, or semicircle shaped; and / or contains one or two protrusions. In some embodiments, each protrusion is from about 3 and about 8 pm. In some embodiments, each protrusion is about 5.25 pm. In some embodiments, the one or two protrusions of each post are substantially parallel to the row including the post. In some embodiments, the array of posts may have a third distance between two adjacent protrusions of two adjacent posts of a row. In some embodiments, the third distance is from about 8 pm to about 14 pm. In some embodiments, the third distance is about 11 pm.

[0017] In some embodiments of any of the foregoing devices, the channel may be a bottom surface, wherein the bottom surface may be an optically transparent region. In some embodiments, the optically transparent region may additionally feature the array of posts. In some embodiments, the channel is fluidically sealed. In some embodiments, the channel may have a top cover with an optically transparent cover region. In some embodiments, the optically transparent cover region may additionally feature the array of posts. In some embodiments, the top cover may be plastic, glass, or quartz.

[0018] In some embodiments, each post may be a height from about 25 pm to about 100 pm. In some embodiments, each post may be a height of about 38 pm. In some embodiments, each post may be a length of from about 12 pm to about 20 pm. In some embodiments, each post may be a length of about 14 pm.

[0019] In some embodiments of the aforementioned devices, each row of the array of posts may be one or more sets of six posts, wherein each set of six posts may have two flanking posts (FP) and four inner posts (IP), arranged in the following configuration FP-IP-IP-IP-IP-FP in the row including the set of six posts. In some embodiments, wherein each FP has one protrusion and each IP has one or two protrusions. In some embodiments, the distance between two adjacent FPs of two adjacent sets of six posts is the first distance; the distance between two adjacent posts of each set of six posts is the second distance; and / or the distance between two protrusions of two adjacent posts of each set of six posts is the third distance. In some embodiments, the channel is in fluidic communication with an outlet. In some embodiments, the inlet is in fluid communication with one or more reagent reservoirs. In some embodiments, the one or more reagent reservoirs may contain a sample reservoir, a reagent reservoir; and / or a buffer reservoir.

[0020] In another aspect, the disclosure features a method of that may involve use of any aforementioned devices and flowing a sample liquid from the inlet through the channel, wherein the sample liquid may be a target cell. In some embodiments, the target cell is smaller than the first distance and larger than the second distance; or the target cell is smaller than the first distance, smaller than the second distance, and larger than the third distance. In some embodiments, the method may additionally involve flowing a first reagent liquid from the inlet through the channel, wherein the first reagent liquid may be a primary labeling agent. In some embodiments, the primary labeling agent is capable of specific binding to the target cell. In some embodiments, the primary labeling agent may be a primary antibody or a small molecule, and the primary labeling agent may be capable of specific binding to a target on the target cell.

[0021] In some embodiments, the primary antibody may be an anti-EpCAM antibody, an anti-EGFR antibody, an anti-MUC1 antibody, an anti-MARS1 antibody, an anti-HER2 antibody, anti-EpCAM antibody, an anti-ER antibody; an anti-PR antibody, an anti-Ki67 antibody, an anti-CD45 antibody, an anti-p53 antibody; an anti-ALK antibody, or a combination thereof. In some embodiments, the small molecule may be 4',6-diamidino-2-phenylindole (DAPI). In some embodiments, the primary labeling agent may be a detection moiety and / or a first nucleic acid. In some embodiments, the detection moiety is a fluorophore.

[0022] In some embodiments, any of the aforementioned methods may further involve flowing a second reagent liquid from the inlet through the channel, wherein the second reagent liquid may be a secondary labeling agent. In some embodiments, the secondary labeling agent is capable of specific binding to the primary labeling agent. In some embodiments, the labeling agent may be a secondary antibody capable of binding to one or more of the primary antibodies and / or a second nucleic acid capable of hybridizing to the first nucleic acid. In some embodiments, the labeling agent may be a detection moiety. In some embodiments, the detection moiety is a fluorophore. In some embodiments, the method disclosed further may involve flowing a wash buffer from the inlet through the channel. In some embodiments, the wash buffer may be a salt, a surfactant, bovine serum albumin, or a combination thereof. In some embodiments, the salt is monosodium phosphate, disodium phosphate, trisodium phosphate, or a combination thereof. In some embodiments, the surfactant is polysorbate 20.

[0023] In some embodiments, the method may further involve flowing an imaging buffer from the inlet through the channel. In some embodiments, the sample liquid, the first reagent liquid, the second reagent liquid, the wash buffer, and / or the imaging buffer are flown into the channel sequentially. In some embodiments, any of the sample liquid, the first reagent liquid, the second reagent liquid, and / or the wash buffer remain in the channel for a duration of time from about 5 seconds to about 30 minutes. In some embodiments, the flowing is at a flow rate of from about 5 pL / min to about 500 pL / min. In some embodiments, the flow rate is about 80 pL / min. In some embodiments, the flowing is for a duration from about 5 seconds to about 30 minutes. In some embodiments, the sample liquid further may additionally contain other particles.

[0024] In some embodiments, the other particles may be cell debris, cell components, inorganic material, non-target cells, or a combination thereof. In some embodiments, non-target cells may be blood cells, immune cells, fibrotic cells, or a combination thereof. In some embodiments, the other particles are smaller than the first distance and the second distance; or smaller than the first distance, the second distance, and the third distance.

[0025] Some embodiments may further involve obtaining one or more images of the channel. In some embodiments, the one or more images are obtained after flowing the sample liquid and the first reagent liquid through the channel; or the sample liquid, the first reagent liquid, and the second reagent liquid through the channel. In some embodiments, the one or more images are obtained at a wavelength corresponding to one or more of the fluorophores on the primary labeling agent and / or the secondary labeling agent. Some embodiments may further involve analyzing the one or more images to identify the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is an epithelial cell, a B cell, a T cell, or a glandular cell.

[0026] In some embodiments, any of the disclosed methods may be used to diagnose a disease; for example, when analyzing a paucicellular specimen. In some embodiments, the disease is a cancer. In some embodiments, the cancer is cholangiocarcinoma, pancreatic cancer, thyroid cancer, liver cancer, lung cancer, lymphoma, head and neck cancer, or breast cancer; and / or metastatic cancer. In some embodiments, the target cell was obtained from a subject using fine-needle aspiration (FNA), cytology brushing, swabbing, a blood sample, or a biopsy sample.

[0027] In some embodiments, a system suitable for use may be used with any one of the aforementioned methods. In some embodiments, a system may involve the device of any one of previously mentioned methods operatively coupled with one or more of: a fluidics control, a microscope, a light source, an optical sensor, a waste container; and / or a computer. In some embodiments, for any of the disclosed systems above, may further involve a sample liquid including a target cell, a first reagent liquid including a primary labeling agent, a second reagent liquid including a secondary labeling agent, a wash buffer; and / or an imaging buffer. In some embodiments, for any of the disclosed systems above, the fluidics control may be a pressure controller, a flow rate sensor, a flow switch controller, and / or a reagent splitter. In some embodiments, the pressure controller may utilize a syringe pump, a peristaltic pump, a pressure-driven pump, or a micro-diaphragm pump. In some embodiments, the fluidics control is operatively coupled with the device to flow a sample liquid, a first reagent liquid, a second reagent liquid, a wash buffer, and / or an imaging buffer into the channel. In some embodiments, the light source may be a light-emitting diode (LED) or one or more lasers. In some embodiments, the light source is capable of emitting a light capable of inducing fluorescence of a fluorophore present on a primary labeling agent and / or a secondary labeling agent. In some embodiments, the optical sensor may be a charge-coupled device (CCD), a photomultiplier tube (PMT), a photodiode, or a complementary metal-oxide- semiconductor (CMOS) device. In some embodiments, the microscope, the light source, and the optical sensor are operatively coupled to the device to obtain one or more images from the channel. In some embodiments, the computer is operatively coupled to the fluidics control, the microscope the light source, and / or the optical sensor. In some embodiments, the computer is programmed to accept as input data one or more images obtained using the system and provide as output data a determination that the target cells may be cancer cells.

[0028] In some embodiments, the disclosure features a computer programmed to accept as input data one or more images obtained using the previously disclosed methods and provide as output data a determination that the target cells may be cancer cells. In some embodiments, the input data may be one or more images obtained using one or more primary and / or secondary labeling agents and one or more images obtained using DAPI, wherein computer determines the position of target cells using the one or more DAPI images, and wherein the computer provides as output data a determination that the target cancer cells may be cancer cells using the one or more images obtained using the one or more primary and / or secondary labeling agents.

[0029] The invention is advantageous when analyzing paucicellular samples which typically may represent a diagnostic challenge. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0030] Brief Description of the Drawings

[0031] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0032] FIG. 1 shows a schematic illustration of an integrated multi-channel automatic profiling (iMAP) system automatic diagnosis system for breast cancer.

[0033] FIG. 2 shows a schematic illustration of an iMAP system for rapid cholangiocarcinoma (CCA) diagnosis. Step 1 : Cytology samples are collected from patients with suspected CCA using bile duct brushing and loaded into a processing solution containing cancer-specific markers (CCAsig and MARS1 ). Step 2: The sample is introduced into an automated microfluidic immunoassay system, where a fluidic control system regulates the flow of reagents. The microfluidic chip captures target cancer cells, immunostains them with specific antibodies, and performs washing steps to remove non-specific debris. Step 3: The immunostained cells are rapidly imaged using fluorescence microscopy to detect cancer biomarkers. Step 4: The captured fluorescence images are analyzed using Cell Profiler software, where computational processing enables automated identification and classification of target cells. Quantitative data, including biomarker intensity and cell counting ratios, are used for diagnosis. The system determines the percentage of normal versus cancerous cells, providing a diagnostic result (positive or negative) within 30 minutes. FIG. 2 abbreviations: iMAP, integrated multi-channel automatic profiling; CCA, cholangiocarcinoma; CCAsig, cocktail of antibodies against EpCAM, EGFR, and MUC1 ; MARS1 , Methionyl-tRNA Synthetase 1 .

[0034] FIG. 3 shows a procedure of cell capture and imaging process in an iMAP system.

[0035] FIG. 4 shows an exemplary design and validation of a single-cell capture in an iMAP system. (A) Schematic illustration of micro-fluidic pattern for specific capture and immunostaining of CCA. (B) Images of iMAP before / after cell capturing and before / after immunostaining. (C) Comparison of the number of captured cells before and after immunostaining. (D) The number of captured cells and cell capturing efficiency depending on the cell counts. (E) The percentage (%) of CCAsig-positive cells depending on the ratio of SNU308 and NIH / 3T3. FIG. 4 abbreviations: CCA, cholangiocarcinoma; iMAP, integrated multichannel automatic profiling system.

[0036] FIG. 5 shows characterization of a microfluidic chip for cancer cell capture in an iMAP system. (A) Microscopic image of the microfluidic chip, highlighting the capture area (red) where target cancer cells are selectively retained and the passing area (blue) where non-target cells and debris flow through. (B) SEM image of the micropatterned structures on the chip, showing precise geometric dimensions optimized for cell trapping. (C) Schematic illustration of the microfluidic chip's function, demonstrating the mechanism of cancer cell capture based on microstructure-induced flow dynamics. The target cells are selectively trapped on the patterned features, while non-specific cells and debris are removed. (D) Comparison of captured cancer cells under static conditions without flow and dynamic conditions with flow in iMAP. FIG. 5 abbreviations: SEM, Scanning electron microscope.

[0037] FIG. 6 shows fluorescence microscopic images of captured CCA cells in a microfluidic chip. (A) DAPI-stained nuclei (blue) of captured cells, showing alignment within the microfluidic trapping structures. (B) Immunofluorescent staining of the same captured cells with CCAsig (green) and MARS1 (red), highlighting the expression of CCA-specific biomarkers. During the staining process, some cells initially captured on the micropattern may detach and be lost, leading to a slight reduction in the total number of stained cells. Despite this minor loss, the microfluidic system effectively retains and labels the majority of target cells for analysis. FIG. 6 abbreviations: CCA, cholangiocarcinoma; CCAsig, cocktail of antibodies against EpCAM, EGFR, and MUC1 ; MARS1 , Methionyl-tRNA Synthetase 1 .

[0038] FIG. 7 shows an overview of a fluidic system and imaging system. (A) BT474 breast cancer cells were captured and imaged by an iMAP system, showing the multichannel imaging capability with one bright field (BF) and four fluorescence channels (DAPI at 405 nm, AF488 at 488 nm channel, AF555 at 555 nm channel, and AF647 at 647 nm channel). (B). The schematic illustration of an iMAP system, including an imaging system, a microfluidic chamber, a fluidic controller system, and a computer that connects the individual components and operates the system.

[0039] FIG. 8 shows an exemplary iMAP optical system. The two photographs on the top show the fabricated imaging system. The CAD and optical schematic show how the optical system was designed in an epifluorescence configuration using a single multi-bandpass filter in front of a tube lens and multiple LEDs coupled with excitation filters. The graph and table on the right show the excitation and emission spectra of the filter sets.

[0040] FIG. 9 shows an illumination simulation employing Zemax. The different lengths between condensing lens and LED (R1 ) and between focusing and condensing lenses (R2) were tested to identify the optimal distance lengths to achieve the maximum illumination power and uniformity.

[0041] FIG. 10 shows an illumination simulation employing Zemax. Light sources with a single LED and four LEDs were compared. The single LED is located in the center of the light source while four LEDs were off-centered in a 2-by-2 configuration. The result shows the light source with four LEDs show 3- times bright light intensity power.

[0042] FIG. 11 shows a comparison of CytoPAN2 and CytoPAN3 image quality. CytoPAN3 refers to an imaging system used in an iMAP system, while CytoPAN2 was a previously reported system (Adv Sci (Weinh). 2022;9(20):e2200415). The background signals and signal-to-noise ratios (SNRs) were compared between the two systems using microbeads coated with AF488 and AF647 fluorescence dyes. The results show the improvement of SNR in the CytoPAN3 approximately by 20-fold.

[0043] FIG. 12 shows a comparison of image quality. Color (Sony IMX250LQR) and monochromatic CMOS camera (Sony IMX250LLR) sensors were compared for an iMAP imaging system. Both cameras used the same Sony IMX250 image sensor. The result shows the color CMOS showed better SNR in green and red channels compared to the monochromatic camera, likely due to lower background noise with an RGB filter implemented in the color CMOS camera.

[0044] FIG. 13 shows spectral unmixing. (A) When using a multi-bandpass filter with multiple excitation LED light sources, fluorescence signals often bleed through adjacent channels. (B) This bleed-through can be corrected by unmixing the fluorescence signals by calculating the ratio of bleed-through for each dye to other channels. (C) The matrix calculation, showing the unmixing method. The system matrix (S, 12 x 5) multiplied by the true signals (T, 5x1 ) is recorded in the image camera (C, 12 x 1 ). From the unmixing calculation, the true signals can be calculated by T = C X S1.

[0045] FIG. 14 shows an exemplary fluidic chip design. The left shows the mask design to fabricate 24 fluidic chips on a 4 inch silicon wafer. The middle shows a single fluidic chip design. The right shows a zoomed-in image in the area of cell capture.

[0046] FIG. 15 shows an exemplary graphical user interface. The left is a microscope GUI that controls an imaging system of iMAP. The right is a fluidics GUI that controls fluidic sequences, flow rates, and time duration.

[0047] FIG. 16 shows validation for a fluidic system. (A) The design of cell capture posts for breast cancer cells (B) The capture efficiency for MCF7 breast cancer cells and PBMC mixtures in different ratios. (C) representative images PBCM labeled by AF555 (orange) and MC7 labeled by AF647 (red) captured on the cell capture area. While MCF7 breast cancer cells were well captured in the capture area, most PBMC passed through. (D) Secondary antibody staining time, showing 10 min is sufficient for cellular immunostaining. (E) Quantification of fluorescent intensity for secondary antibody staining time, shown in (D).

[0048] FIG. 17 shows iMAP characterization of CCA cell lines. (A) Representative fluorescence images of NIH / 3T3 (non-cancer) and SNU308 (CCA) cells stained against DAPI, CCAsig, and MARS1 . (B-D) Histogram of fluorescence intensity from iMAP analysis for CCAsig (C) and MARS1 (D) markers. Three CCA cell lines (SNU308, SNU478, SNU869) and control cell lines (NIH / 3T3) were used. The dashed line indicates the threshold value that determines the marker positivity. (E) Median fluorescence intensity of CCAsig and MARS1 for CCA and control cell lines. (F) Marker-positive cell percentage of CCAsig and MARS1 for CCA and control cell lines. FIG. 17 abbreviations: CCA, cholangiocarcinoma; CCAsig, cocktail of antibodies against EpCAM, EGFR, and MUC1 ; DAPI, 4’,6’-diamidino-2-phenylindole; MARS1 , Methionyl-tRNA Synthetase 1 .

[0049] FIG. 18 shows validation for a fluidic system. 10 min antibody incubation showed the highest signal-to-noise ratio difference between BT474 (positive sample for QUAD, ER / PR, and HER2) and Jurkat (negative sample for QUAD, ER / PR, and HER2). FIG. 18 abbreviations: Quad, cocktail of antibodies against ER, PR, Ki67, CD45 (negative marker)); ER, estrogen; PR, progesterone. FIG. 19 shows fluorescence microscopic images of immunostained NIH / 3T3, SNU478, SNU869, and SNU308 cells captured on a microfluidic chip. Histograms and cut-off levels of NIH3T3, SNU478, SNU869, and SNU308 cell lines after immunostaining with IgG 1 and lgG2a antibodies (see FIG. 20). NIH / 3T3 (negative control) and CCA cell lines (SNU478, SNU869, and SNU308) were immunostained with DAPI (blue, nucleus staining), CCAsig (green) and MARS1 (red). The images show selective binding of CCAsig and MARS1 in the CCA cell lines, with minimal signal in the NIH / 3T3 control group, validating the specificity of the biomarkers. The microfluidic chip effectively captures and aligns cells for high- throughput fluorescence imaging. Scale bar: 100 pm. FIG. 19 abbreviations: CCA, cholangiocarcinoma; CCAsig, cocktail of antibodies against EpCAM, EGFR, and MUC1 ; DAPI, 4’,6’-diamidino-2-phenylindole; MARS1 , Methionyl-tRNA Synthetase 1 .

[0050] FIG. 20 shows histograms of background signals for NIH3T3, SNU478, SNU869, and SNU308 cell lines after immunostaining with (A) IgG 1 and (B) lgG2a antibodies.

[0051] FIG. 21 shows validation for a fluidic system. (A) Cellular fluorescence images using CytoPAN3 (an imaging system for iMAP) for breast cancer cell lines (BT474, MCF7, MDAMB231 , SKBR3) and negative control cell line (Jurkat). (B) Fluorescence intensities of cells for ER / PR, HER2, and QUAD markers. (C). Comparison of marker profiling using conventional Zeiss microscope and CytoPAN3 system. (D) Correlation of fluorescence intensities between Zeiss and CytoPAN3 systems for ER / PR, HER2, and QUAD markers in four different breast cancer cell lines. FIG. 21 abbreviations: Quad, cocktail of antibodies against ER, PR, Ki67, CD45 (negative marker)); ER, estrogen; PR, progesterone.

[0052] FIG. 22 shows an exemplary heatmap representation of Z-scores for CCAsig and MARS1 using iMAP (green) and flow cytometry (FC, red). FIG. 22 abbreviations: CCA, cholangiocarcinoma; CCAsig, cocktail of antibodies against EpCAM, EGFR, and MUC1 ; MARS1 , Methionyl-tRNA Synthetase 1 .

[0053] FIG. 23 shows CCA diagnosis analysis. (A) Heatmap of all patient samples from each marker: CCAsig and MARS1 (the color scale indicates the number of positive cells). Comparison for (B) the number of total captured cells, (C) the number of CCAsig positive cells, (D) the number of MARS1 positive cells, and (E) the number of CCAsig or MARS1 positive cells between CCA and benign (Mann-Whitney t- test). (F) A waterfall plot of CCA and benign for the number of CCAsig or MARS1 positive cells. (G) Receiver operating characteristic (ROC) curves of CCAsig-positive (AUC = 0.80), MARS1 -positive cells (AUC = 0.74), and CCAsig or MARS1 -positive cells (AUC = 0.82). FIG. 23 abbreviations: CCA, cholangiocarcinoma; CCAsig, cocktail of antibodies against EpCAM, EGFR, and MUC1 ; DAPI, 4’, 6’- diamidino-2-phenylindole; MARS1 , Methionyl-tRNA Synthetase 1 .

[0054] FIG. 24 shows bio-marker expression in FNA clinical samples for (A) QUAD, (B) ER / PR, and (C) HER2. (D) The difference of QUAD-positive cells between malignant and benign samples. (E) AUROC of differentiating malignant from benign using QUAD-positive cell counts was 0.8438. (F) ER / PR-positive cell counts in ER / PR-positive or negative samples. (G) ER / PR-positive cell counts for samples negative for both markers, positive for either marker, or positive for both markers. (H) HER2-positive cell counts for samples negative or positive for HER2. (I) HER2-positive cell counts based on HER2 positivity. FIG. 24 abbreviations: Quad, cocktail of antibodies against ER, PR, Ki67, CD45 (negative marker)); ER, estrogen; PR, progesterone.

[0055] FIG. 25 shows Quad marker expression in cells from FNA clinical samples. Total captured cell counts, QUAD-positive cell counts, and their percentages were measured and compared between malignant and benign cases. The area under the ROC curve (AUROC) to differentiate between malignant and benign was 0.9286. FIG. 25 abbreviations: Quad, cocktail of antibodies against ER, PR, Ki67, CD45 (negative marker)); ER, estrogen; PR, progesterone.

[0056] FIG. 26 shows Quad marker expression of PE clinical samples. Total captured cell counts, QUAD-positive cell counts, and their percentages were measured.

[0057] FIG. 27 shows Quad marker expression of CSF clinical samples. Total captured cell counts, QUAD-positive cell counts, and their percentages were measured. Representative fluorescence images are shown.

[0058] FIG. 28 shows Quad marker expression in all clinical sample types (FNA, CSF, PE). QUAD- positive cell count percentages were measured and compared between malignant and benign cases. The AUROC to differentiate between malignant and benign was 0.96.

[0059] FIG. 29 shows the top-down views of three types of post geometries. The views include two posts on the flanks of their respective capture areas with a passing area in between. First, second, and third distances are annotated for each type of post geometry. A: T-shaped posts flanked by two L-shaped posts; B: triangular posts; and C: rounded posts.

[0060] Definitions

[0061] As used herein, the term “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre- ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood). A biological sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A biological sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule. A biological sample may also refer to intact cells (e.g., imaged in 2D or 3D). A biological sample may refer to an in vitro cell or in vitro cells (e.g., cultured cells or cultured cell lines).

[0062] In any of the methods described herein, the biological sample may include a cell, a tissue, and / or a cellular component. The biological sample may have been obtained from a human. The biological sample may have been obtained from a subject with a disorder (e.g., a cancer; e.g., a breast cancer). The disorder may also be an infectious disease (e.g., resulting from a bacteria, virus, fungus, or parasite), a cardiovascular disease, a respiratory disease, a metabolic disorder, an endocrine disorder, a neurological disorder, a mental disorder, an autoimmune disease, a gastrointestinal disease, a rare disease. The biological sample may include human cancer cells. The biological sample may include human breast cancer carcinoma cells. In some instances, the biological sample includes an in vitro cell sample. The cell sample may include one or more cell lines (e.g., one or more cell lines suitable for modeling a disorder). As used herein, the term “optically transparent” refers to materials that allow light to pass through. In some instances, light is able to pass through optically transparent materials without appreciable scattering, which is measured by total light transmittance. In some instances light transmittance of an optically transparent material may be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0063] As used herein, the term “substantially perpendicular” refers to objects, groups of objects (e.g. rows of posts), or directions that are 90 degrees in relationship to one another within the range of about + / - 10%, about + / - 9%, about + / - 8%, about + / - 7%, about + / - 6%, about + / - 5%, about + / - 5%, about + / - 4%, about + / - 3%, about + / - 2%, about + / - 1%. As used herein, the term “substantially parallel” refers to two or more objects, groups of objects, or two directions that lie 0 or 180 degrees in reference to one another within the range of about + / - 10%, about + / - 9%, about + / - 8%, about + / - 7%, about + / - 6%, about + / - 5%, about + / - 5%, about + / - 4%, about + / - 3%, about + / - 2%, about + / - 1%.

[0064] As used herein, the term “capable of specific binding” refers to molecule that can bind specifically to a compound of interest in a heterogenous mixture, for instance in a biological sample. As used herein, the term “primary labeling agent” refers to a molecule that binds specifically to a compound of interest (e.g. a surface protein) in a complex heterogenous mixture, for instance in a biological fluid or sample. Primary labeling agents may be conjugated to fluorophores, enzymes, or other molecules (e.g. streptavidin or biotin) that allow for them to be directly visualized within a complex heterogenous mixture. As used herein, the term “secondary labeling agent” refers to a molecule that binds specifically to the primary labeling agent (e.g. primary antibody) in a complex heterogenous mixture, for instance in a biological fluid or sample. Secondary labeling agents may be conjugated to fluorophores, enzymes, or other molecules (e.g. streptavidin or biotin) that allow for them to be directly visualized within a complex heterogenous mixture.

[0065] As used herein, the term “small molecule” refers to a molecule with a molecular weight at or below 1000 Daltons (Da). Small molecules typically exclude proteins (e.g., antibodies) and nucleic acids (e.g., oligonucleotides and polynucleotides).

[0066] As used herein, the term “computer” refers to a programmable device that performs a wide range of tasks by processing data. A typical computer may have core functions that include storing, retrieving, and processing information.

[0067] Detailed Description

[0068] The present invention relates to devices, methods, and systems for analyzing a biological sample (e.g., including target cells). In particular, the devices described herein include microfluidic devices for manipulating, staining, imaging, and / or analyzing target ceils to determine whether the target cells are indicative of a disease, e.g., a cancer, as described herein, and / or whether a subject from whom the target ceils are obtained has a disease, e.g., a cancer, as described herein. The methods described herein include use of the device to perform an in vitro diagnostic method to manipulate, stain, image, and / or analyze target cells to determine whether the target cells are indicative of a disease, e.g., a cancer, as described herein, and / or whether a subject from whom the target cells are obtained has a disease, e.g., a cancer, as described herein. The methods may further include flowing a sample liquid including the target cells through a device herein, wherein the target ceils are immobilized in the device (e.g., in a region of the device which may be imaged): labeling the target celis with one or more primary and / or secondary labeiing agents described herein by e.g., flowing a reagent liquid and / or a wash buffer; and / or imaging the labeled target cells, e.g., using fluorescence microscopy. The systems described herein include the devices described herein, and are suitable for use with any of the methods described herein. The systems may further include one or more reagent reservoirs, a fluidics control, a microscope, a light source, an optical sensor, a waste container, and / or a computer.

[0069] Below we describe methods and systems to enrich and molecularly analyze paucicellular tumor cells in cytology specimens (e.g., fine-needle aspiration, brushing, cerebrospinal fluids, pleural fluids) that are difficult to form tissue blocks and analyze by immunohistochemistry. These methods, for example, enable rapid immunolabeling and detection of tumor cells to improve cancer diagnosis accuracy and molecular phenotyping. The technology, in general, consists of a device for sample processing (sample preprocessing, target enrichment, and immunolabeling) and a portable imaging system for operation and read-out at a point-of-care setting.

[0070] I. Microfluidic Device

[0071] Described here are microfluidic devices including an inlet in fluid communication with a channel. The channel may include an array containing one or more rows of posts, wherein each row of posts includes one or more capture areas and one or more passing areas, wherein the one or more capture areas includes the posts and the passing area does not include any posts.

[0072] A. Inlet

[0073] The microfluidic devices described herein include an outlet in fluid communication with a channel. In the method described herein, flow of one or more liquids (e.g., a sample liquid, a reagent liquid, a wash buffer, or an imaging buffer) may be directed from the inlet into the channel. Devices and systems described herein may further include one or more reservoirs (e.g., for holding a sample liquid, a reagent liquid, a wash buffer, or an imaging buffer) which may be in fluid communication with the inlet.

[0074] The inlet may be of any dimension (e.g., with a square, rectangular, oval, elliptical, rounded, or circular cross-section) and shape suitable for the microfluidic device and suitable for being in fluid communication with the channel. For example, the inlet may have an inner dimension (e.g., a height or a width) of about 0.5, 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 75, 100, 200, 250, 500, 750, or 1000 pm.

[0075] B. Channel

[0076] The microfluidic devices described herein typically include a channel including an array of posts. The array of posts may include one or more passing areas and one or more capture areas.

[0077] The passing areas may be structured such that in any of the methods described below, when flowing a sample liquid (e.g., including a target cell and / or other particles) or other liquid through the device (e.g., the channel), everything (e.g., including the liquid, the target cell, and the other particles) may freely pass through the passing area, e.g., towards a subsequent row of posts in the array, relative to the direction of flow.

[0078] The capture areas may be structured such that in any of the methods below, when flowing a sample liquid (e.g., including a target cell and / or other particles) or other liquid through the device (e.g., the channel), the target cell is captured (i.e., immobilized) within a capture area. In particular, the capture area may include posts (described in detail below), which may be structured in a way such that the target cell cannot pass through the capture area and is captured (e.g., immobilized) within. In particular, the target cell may be captured by the posts within the capture area, e.g., because the target cell is larger than the distance between the posts. As described in further detail below, the posts in the capture area may be further structured such that the space between adjacent posts contains a narrowing or funneling shape (e.g., a larger distance between two adjacent posts within a capture area in the direction against the direction of flow and a smaller distance between two adjacent posts within a capture area in the direction along the direction of flow).

[0079] In some instances, the array (of posts) may include at least two rows of posts, wherein a first row of posts is adjacent to a second row of posts, and wherein the geometric center of a first capture area in the first row does not align with the geometric center of any capture area in the second row along a direction substantially perpendicular to the rows; and / or the geometric center of a first passing area in the first row does not align with the geometric center of any passing area in the second row along a direction substantially perpendicular to the rows. In the methods described within, when flowing a sample liquid through the channel, a target cell which passes through a passing area of a first row may be directed towards the capture area of a subsequent row in this manner.

[0080] In some of the microfluidic devices described herein, each row may include 1 to 50 capture areas (e.g., 1 capture area, 2 capture areas, 3 capture areas, 4 capture areas, 5 capture areas, 6 capture areas, 7 capture area, 8 capture areas, about 10 capture area, about 20 capture areas, about 30 capture area, about 40 capture areas, about 45 capture areas, or 50 capture areas), and 1 to 50 passing areas (e.g., 1 passing area, 2 passing areas, 3 passing areas, 4 passing areas, 5 passing areas, 6 passing areas, 7 passing area, 8 passing areas, about 10 passing area, about 20 passing areas, about 30 passing area, about 40 passing areas, about 45 passing areas, or 50 passing areas). In some of the devices described herein, each capture area may include two to ten posts (e.g., 2 posts, 3 posts, 4 posts, 5 posts, 6 posts, 7 posts, 8 posts, 9 posts, or 10 posts).

[0081] In some of the microfluidic devices described herein, the microfluidic device may include fifty to five hundred rows (e.g., about 50 rows, about 55 rows, about 60 rows, about 65 rows, about 70 rows, about 80 rows, about 100 rows, about 150 rows, about 200 rows, about 250 rows, about 300 rows, about 350 rows, about 400 rows, or about 500 rows). In some of the microfluidic devices described herein, the rows are substantially parallel.

[0082] In some of the microfluidic devices described herein, each passing area may be larger in size in a direction parallel to the row including the passing area than the distance between any two adjacent posts in a single capture area. In some instances, the passing areas, which can also be defined by the distance between two adjacent posts in two adjacent capture areas (e.g., FIG. 29), may be larger than the distance any two adjacent posts in a single capture area, particularly for passing areas and capture areas within the same row of posts. This size may be described as a first distance in the present disclosure.

[0083] In some of the microfluidic devices described herein, the size of a passing area in a direction parallel to the row including the passing area of a first row of posts is different from the size of a passing area in a direction parallel to the row including the passing area of a second row of posts. For example, the size of the passing area may be larger (e.g., wider) in rows closer to inlet (e.g., FIG. 29). These two sizes may be described as a second distance and a third distance, respectively in the present disclosure.

[0084] In some of the microfluidic devices described herein, the distance between any two adjacent posts in a single capture area of a first row of posts is different from the distance between any two adjacent posts in a single capture area of a second row of posts. For example, any two adjacent posts in a single capture area may be larger (e.g., wider) in rows closer to inlet).

[0085] In some instances, the size of a passing area in a direction substantially parallel to the row including the passing area is a first distance and the distance between any two adjacent posts in a single capture area is a second distance.

[0086] A device described herein may include an inlet; and a channel in fluidic communication with the inlet, wherein the channel includes an array of posts positioned in one or more rows with a first distance and a second distance between adjacent posts in each row.

[0087] In some of the microfluidic devices described herein, the first distance is at least 20 pm (e.g., about 20 pm, about 22.5 pm, about 27.5 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, or more). In some of the microfluidic devices described herein, the first distance is about 45 pm.

[0088] In some of the microfluidic devices described herein, the second distance is from about 15 pm to about 25 pm (e.g. about 15 pm, about 17.5 pm, about 20 pm, about 22.5 pm, or about 25 pm). In some of the microfluidic devices described herein, the second distance is about 22.5 pm.

[0089] In some of the microfluidic devices described herein, the channel may include a bottom surface, wherein the bottom surface includes an optically transparent region. In some instances the optically transparent region of the channel includes the array of posts.

[0090] In some of the microfluidic devices described herein, the channel is flu idically sealed.

[0091] In some of the microfluidic devices described herein, the channel includes a top cover. In some instances, the top cover includes an optically transparent cover region. The top cover may be fluidically sealed or completely sealed to the channel. The top cover may be detachable or be part of the channel. In some instances the optically transparent cover region includes the array of posts. In some instances, the top cover includes plastic, glass, or quartz. In some instances, the presence of the top cover is optional (e.g., the channel may be open).

[0092] In some instances, the top cover may not be optically transparent, e.g., may not include an optically transparent cover region. In such instances, the bottom surface should include optically transparent region.

[0093] In some of the microfluidic devices described herein, the channel is in fluidic communication with an outlet.

[0094] In some of the microfluidic devices described herein, the inlet is in fluid communication with one or more (e.g., 1 reagent reservoir, 2 reagent reservoirs, 3 reagent reservoirs, 4 reagent reservoirs, 5 reagent reservoirs, 6 reagent reservoirs, 7 reagent reservoirs, 8 reagent reservoirs, about 10 reagent reservoirs, or more) reagent reservoirs. In some instances, the one or more (e.g., 1 reagent reservoir, 2 reagent reservoirs, 3 reagent reservoirs, 4 reagent reservoirs, 5 reagent reservoirs, 6 reagent reservoirs, 7 reagent reservoirs, 8 reagent reservoirs, about 10 reagent reservoirs, or more) reagent reservoirs may include: a sample reservoir; a reagent reservoir; and / or a buffer reservoir. C. Posts

[0095] Some of the devices described herein may include posts. In some instances, two adjacent posts within a capture area may form a “pocket” or a “capture site.”

[0096] Posts may be any suitable shape or dimension suitable for a device which can be used with the methods described herein, and multiple posts may be positioned within a capture area in a manner suitable for a device which can be used with the methods described herein.

[0097] In some of the microfluidic devices described herein, each post: is T-shaped (e.g., FIG. 29), L- shaped (e.g., FIG. 29), triangular (e.g., FIG. 29), or rounded (e.g., FIG. 29); and / or contains one (e.g., L- shaped; e.g., FIG. 29) or two protrusions (e.g., T-shaped; e.g., FIG. 29). In some instances, each protrusion is from about 3 and about 8 pm pm (e.g. about 3 pm, about 3.5 pm, about 4 pm, about 4.5 pm, about 5 pm, about 5.5 pm, about 6 pm, about 6.5 pm, about 7 pm, about 7.5 pm, or about 8 pm). In some instances, each protrusion is about 5.25 pm. In some instances, the one or two protrusions of each post are substantially parallel to the row including the post (e.g., FIG. 29).

[0098] In some of the microfluidic devices described herein, the array of posts includes a third distance between two adjacent protrusions of two adjacent posts of a row (e.g., FIG. 29). In some instances, the third distance is from about 8 pm to about 14 pm (e.g. about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm). In some instances, the third distance is about 11 pm.

[0099] In some of the microfluidic devices described herein, each post may have a height from about 25 pm to about 100 pm (e.g. about 25 pm, about 27.5 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm). In some instances, each post may have a height of about 38 pm. The height of the post may depend on the fabrication method of the microfluidic device (e.g., may depend on the resist for a microfluidic device fabricated by lithography). The height may refer to a vertical distance substantially perpendicular to the bottom surface of the channel.

[0100] In some of the microfluidic devices described herein, each post may have a length of from about 12 pm to about 20 pm (e.g. about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 17.5 pm, about 18 pm, or about 20 pm). In some instances, each post may have a length of about 14 pm. The length may refer to a horizontal distance substantially parallel to the direction of flow of the channel (e.g., substantially perpendicular to the row of posts including the post).

[0101] In some of the microfluidic devices described herein, each row of the array of posts includes one or more sets (e.g., 1 set, 2 sets, 3 sets, 4 sets, 5 sets, 6 sets, 7 sets, 8 sets, about 10 sets, about 15 sets, or more) of six posts, wherein each set of six posts includes two flanking posts (FP) and four inner posts (IP), arranged in the following configuration FP-IP-I P-l P-IP-FP in the row containing the set of six posts. In a more general instance, each row of the array of posts may include one or more (e.g., 1 set, 2 sets, 3 sets, 4 sets, 5 sets, 6 sets, 7 sets, 8 sets, about 10 sets, about 15 sets, or more) sets of N posts, including two FP and (N-2) IP, arranged in the following configuration FP-IP-...-IP-FP. In some instances, (e.g., when the posts are T-shaped and L-shaped, each FP has one protrusion (T-shaped) and each IP has two protrusions (L-shaped).

[0102] In some of the microfluidic devices described herein, the distance between two adjacent FPs of two adjacent sets of six posts is the first distance; the distance between two adjacent posts of each set of six posts is the second distance; and / or the distance between two protrusions of two adjacent posts of each set of six posts is the third distance (e.g., FIG. 29).

[0103] A microfluidic device described herein may be fabricated using polydimethylsiloxane (PDMS) using a micro-patterned silicon wafer template. The micro-patterned silicon wafer is prepared by Heidelberg uPG501 (Heidelberg Instruments, Germany) using SU-8. Liquid silicone elastomer and curing agent with a weight ratio of 10:1 are thoroughly mixed. The PDMS mixture is degassed in a vacuum desiccator for 30 min. The PDMS mixture is poured on the micro-patterned silicon wafer and then baked in the drying oven at 60 °C overnight to cure the PDMS. The cured PDMS is cut by a surgical knife along the pattern boundary, then peeled off from the silicon wafer and punched by a 2.5 mm biopsy puncher to make an inlet and outlet. The prepared PDMS and slide glass are treated with O2 plasma for 1 min for plasma bonding. Then, the treated side of each of them is attached and cured at 60 °C for 12 hours.

[0104] An exemplary design for a microfluidic chip described herein is shown in FIG. 14, particularly showing the spacing of the rows of posts within a channel and the spacing between posts.

[0105] II. Biological Samples

[0106] The microfluidic devices described herein may be used to analyze a biological sample, e.g., containing a target cell. The analysis may be performed using any of the methods (e.g., diagnostic methods; e.g., in vitro methods) described herein. The target cell may be associated with a disease, e.g., a cancer, as described below. In such instances, target cell may be a cancer cell, e.g., of the cancer type. A sample fluid described herein (e.g., in the methods described below), may include a biological sample, e.g., a target cell. The biological sample may be obtained from a subject, e.g., a subject suspected to have a disease (e.g., cancer) or to be tested for having a disease (e.g., cancer).

[0107] A. Sample Types

[0108] In some instances, the target cell is a cancer cell. In some instances, the target cell is an epithelial cell, a B cell, a T cell, or a glandular cell. In some instances, the cancer is cholangiocarcinoma, pancreatic cancer, thyroid cancer, liver cancer, lung cancer, lymphoma, head and neck cancer, or breast cancer. In some instances, the cancer is metastatic cancer.

[0109] Exemplary cell lines useful in the methods described herein include SNU308, SNU478, and SNU869. These cell lines may be cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen) supplemented with 10% FBS. Other exemplary cell lines include NIH / 3T3, SNU308, SNU478, and SNU869. NIH / 3T3, which may be grown in Dulbecco’s minimum essential medium (DMEM) (Hyclone) with 10% fetal bovine serum (FBS) (Gibco, Invitrogen). Further exemplary cell lines include MDAMB231 , MCF7, SKBR3, BT474

[0110] B. Sample Collection

[0111] The biological sample, e.g., target cell may be obtained by any of the methods known in the art, e.g., from a subject. In particular, a target cell may be obtained (e.g., from a subject) using fine-needle aspiration (FNA), cytology brushing, swabbing, a blood sample, or a biopsy sample. Preferred methods for collecting a biological sample to be analyzed with the methods described herein include FNA and cytology brushing. III. Diagnostic Methods

[0112] Any of the methods described herein may be used to determine if a target cell is a cancer cell. Alternatively, some instances of the methods described herein may be used to diagnose a disease, e.g., in a subject. In some instances, the disease is a cancer. In some instances, a biological sample including a target cell is obtained from the subject, and some instances of the methods described may be used to determine whether the target cell in the biological sample obtained from the subject is a cancer cell. In some instances, the cancer is cholangiocarcinoma, pancreatic cancer, thyroid cancer, liver cancer, lung cancer, lymphoma, head and neck cancer, or breast cancer. This cancer may additionally be metastatic.

[0113] Any of the methods described may use or be used with any of the microfluidic devices described. The methods may additionally include flowing a sample liquid from the inlet through the channel, wherein the sample liquid includes a target cell. The flowing may be performed according to any methods known in the art or described herein. For any of the methods described, the sample liquid, the first reagent liquid, the second reagent liquid, the wash buffer, and / or the imaging buffer, as well as any other liquids) may be flown into the channel sequentially. For any of the methods described, the sample liquid, the first reagent liquid, the second reagent liquid, the wash buffer, and / or the imaging buffer, as well as any other liquids) may be flown into the channel in any combination thereof.

[0114] A. Cell Filtering

[0115] For any of the methods described, the target cell (e.g., in the sample liquid) may be smaller than the first distance and larger than the second distance; or the target cell may be smaller than the first distance, smaller than the second distance, and larger than the third distance. In addition, the sample liquid may further includes other particles. These particles may be cell debris, cell components, inorganic material, non-target cells, or a combination thereof. The non-target cells may refer to blood cells, immune cells, fibrotic cells, or a combination thereof. The other particles may also be smaller than the first distance and the second distance; or smaller than the first distance, the second distance, and the third distance.

[0116] In the methods described herein using any of the devices described herein, a target cell may flow through one or more passing areas within the channel. In the methods described herein using any of the devices described herein, a target cell may be captured (e.g., immobilized) by a capture area. In particular, the target cell may be captured between two adjacent posts within a capture area (e.g., within a pocket or capture site).

[0117] B. Cell labeling

[0118] For any of the methods described, the first reagent liquid which enters from the inlet through the channel may be a primary labeling agent. This primary labeling agent may be capable of specific binding to the target cell. Optionally, the primary labeling agent may be a primary antibody or a small molecule, that is capable of specific binding to a target on the target cell. Exemplary primary antibodies include an anti-EpCAM antibody, an anti-EGFR antibody, an anti-MUC1 antibody, an anti-MARS1 antibody, an anti- HER2 antibody, anti-EpCAM antibody, an anti-ER antibody; an anti-PR antibody, an anti-Ki67 antibody, an anti-CD45 antibody, an anti-p53 antibody; an anti-ALK antibody, or a combination thereof. An exemplary small molecule that may be used for staining is 4',6-diamidino-2-phenylindole (DAPI). The primary labeling agent may contain a detection moiety and / or a first nucleic acid. The detection moiety can be a fluorophore.

[0119] The second reagent liquid from the inlet may be a secondary labeling agent. In some instances, the secondary labeling agent is capable of specific binding to the primary labeling agent. In some instance, the labeling agent is a secondary antibody capable of binding to one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, about 10, about 12, or more) of the primary antibodies and / or a second nucleic acid capable of hybridizing to the first nucleic acid. The labeling agent may also contain a detection moiety. In some instances, the detection moiety is a fluorophore. In some instances, for any of the methods described, there may also be flowing from a wash buffer from the inlet through the channel. The wash buffer may include a salt, a surfactant, bovine serum albumin, or a combination thereof. In some instances, the salt is monosodium phosphate, disodium phosphate, trisodium phosphate, or a combination thereof. In some instances, the surfactant is polysorbate 20.

[0120] As a non-limiting example for cholangiocarcinoma (CCA), a panel of CCA cell lines with different expressions of CCAsig (EpCAM, EGFR, MUC1 ) and MARS1 markers are used for validation:

[0121] For any of the methods described, the sample liquid, the first reagent liquid, the second reagent liquid, and / or the wash buffer remains in the channel for a duration of time from about 5 seconds to about 30 minutes (e.g. about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 10 seconds, about 12 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 10 minutes, about 12 minutes, about 14 minutes, about 16 minutes, about 20 minutes, about 22.5 minutes, about 25 minutes, about 27.5 minutes, or about 30 minutes). In addition, the flowing occurs at a flow rate of from about 5 pL / min to about 500 pL / min (e.g., about 5 pUmin, about 6 pL / min, about 7 pL / min, about 8 pL / min, about 10 pL / min, about 12 pL / min, about 15 pL / min about 20 pL / min, about 25 pL / min, about 30 pL / min, about 40 pL / min, about 50 pL / min, about 75 pL / min, about 100 pL / min, about 150 pL / min, about 225 pL / min, about 250 pL / min, about 275 pL / min, about 300 pL / min, about 325 pL / min, about 350 pL / min, about 375 pL / min, about 400 pL / min, about 450 pL / min, about 475 pL / min, or about 500 pL / min). In some instances, the flow rate is about 80 pL / min. The flowing occurs for a duration from about 5 seconds to about 30 minutes.

[0122] In an exemplary immunostaining (e.g., immunohistochemistry or IHC) protocol, antibodies are solubilized in PBS mixed with 0.5% tween, 2.5 wt% of BSA for blocking, and 1 X BD Perm buffer for permeabilization. Primary (1st) antibodies and secondary (2nd) antibodies are prepared into cocktail solutions. Cells are incubated in primary antibodies solution for 10 min and put in a microfluidic device described herein (e.g., iMAP). The incubated cell solution is added to capture cells with an 80 pUmin flow rate for 2.75 min. Then, the PBST solution flows with 0.4 bar for 15 sec and 80 pUmin for 4 min to remove unbound primary antibodies. The secondary antibody solution is injected with 30 pL / min for 5 min. Finally, the PBST solution flowed with 80 pL / min for 4 min to push out the excess secondary antibodies.

[0123] In another exemplary method of staining target cells, after trypsinizing NIH / 3T3, H69, SNU308, SNU478, and SNU869 cells, they are washed with PBS and fixed with CytoRich Red (Thermo Scientific, MA, USA) to keep them in 4 °C. The fixed cells are blocked with 0.5% BSA in PBS for 30 min and are incubated with primary antibodies for 1 h. The AF488 anti-mouse secondary antibodies are used to bind the primary antibodies for 30 min. CytoFlex flow cytometer (Beckman Coulter) using the 488 / 8 nm bandpass filter for FSC (48 V), SSC (29 V), and FITC (20 V).

[0124] In some instances, a set of cocktail antibodies (CCAsig) is used against EpCAM, EGFR, and MUC1 in the 475 nm channel (e.g., primary antibodies against EpCAM, EGFR, and MUC1 (CCAsig)) and secondary antibodies containing a fluorophore in the 475 nm channel) and antibodies against MARS1 in the 630 nm channel (e.g., primary antibodies against MARS1 and secondary antibodies containing a fluorophore in the 630 nm channel). DAPI staining is done in the 385 nm channel. Alternatively, CCAsig can be stained in the AF488 channel (e.g., primary antibodies against EpCAM, EGFR, and MUC1 (CCAsig)) and secondary antibodies containing Alexa Fluor 488) and MARS1 can be stained in the AF647 (e.g., primary antibodies against MARS1 and secondary antibodies containing Alexa Fluor 647).

[0125] In some instances, fixed cells are blocked with 0.5% BSA in PBS for 30 min and incubated with primary antibodies for 1 hour. The secondary antibodies are used to bind the primary antibodies for 30 min. For example, if the primary antibody or antibodies (e.g., for the CCAsig cocktail) are mouse antibodies, the secondary antibodies may be anti-mouse antibodies. If the primary antibody or antibodies are rabbit antibodies, the secondary antibodies may be anti-rabbit antibodies. Suitable antibody combinations (e.g., mouse, rabbit, goat, guinea pig, donkey, sheep, chicken, etc.) known in the art may be used for primary / secondary antibody staining described herein.

[0126] C. Imaging

[0127] For any of the methods described, flowing of an imaging buffer from the inlet may occur through the channel. The imaging buffer may remain in the channel for a period of time sufficient to obtain one or more images (e.g., fluorescence images which total 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images) of one or more target cells (e.g., 1 cell, 2 cells, 3 cells, 4 cells, 5 cells, 6 cells, 7 cells, 8 cells, about 10 cells, about 20 cells, about 25 cells, about 30 cells, about 50 cells, about 100 cells, about 200 cells, about 250 cells, about 300 cells, about 400 cells, about 500 cells, about 600 cells, about 700 cells, about 800 cells, about 900 cells, about 1000 cells, about 2000 cells, about 3000 cells, about 5000, or more) captured by the array of one or more posts (e.g. 1 post, 2 posts, 3 posts, 4 posts, 5 posts, 6 posts, 7 posts, 8 posts, about 10 posts, about 12 posts, about 14 posts, about 16 posts, about 18 posts, about 20 posts, about 25 posts, about 30 posts, about 35 posts, about 40 posts, about 45 posts, about 50 posts, about 60 posts, about 70 posts, about 80 posts, about 90 posts, about 100 posts, or more). In some instances, there may be the one or more images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, or about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, 1000 images, or more) obtained after flowing: the sample liquid and the first reagent liquid through the channel; or the sample liquid, the first reagent liquid, and the second reagent liquid through the channel. In some instances, there may be one or more images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, or more) are obtained at a wavelength corresponding to one or more of the fluorophores (e.g., 1 fluorophore, 2 fluorophores, 3 fluorophores, 4 fluorophores, 5 fluorophores, 6 fluorophores, 7 fluorophores, 8 fluorophores, about 10 fluorophores, about 12 fluorophores, about 15 fluorophores, or more) on the primary labeling agent and / or the secondary labeling agent. These images one or more images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, or more) may be analyzed to identify the target cell.

[0128] As a non-limiting example, fluorescence images of captured cells in a microfluidic device described herein (e.g., an iMAP system) are acquired using a Zeiss upright automated epifluorescence microscope. The acquired data is analyzed using CellProfi ler software. All cells were stained with DAPI, CCAsig, and MARS1 . During the image analysis process, DAPI-stained cells were first identified, and their outlines were delineated to define cell boundaries. Based on these identified DAPI-positive cells, fluorescence signal intensities were subsequently measured for CCAsig in the AF488 channel and MARS1 in the AF647 channel to evaluate their respective fluorescence intensities. The signal intensity was then used to determine positivity by applying a cutoff.

[0129] The isotype control was used for the normalization of each cell.

[0130] IV. Systems

[0131] Described herein are systems suitable for use any of the methods described herein. The systems described herein may include any of the microfluidic devices described herein.

[0132] For any of the systems described, the device may be operatively coupled with one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, about 10, about 12, about 14, about 16, about 20, or more) of a fluidics control, a microscope, a light source, an optical sensor, a waste container; and / or a computer. The system may also have a sample liquid including a target cell, a first reagent liquid including a primary labeling agent, a second reagent liquid including a secondary labeling agent, a wash buffer; and / or an imaging buffer.

[0133] A. Fluidics

[0134] For any of the systems described herein, the fluidics control may include a pressure controller, a flow rate sensor, a flow switch controller, and / or a reagent splitter. In some instances, the pressure controller includes a syringe pump, a peristaltic pump, a pressure-driven pump, or a micro-diaphragm pump. In some instances, the fluidics control is operatively coupled with the device to flow a sample liquid, a first reagent liquid, a second reagent liquid, a wash buffer, and / or an imaging buffer into the channel. For example, a ELVEFLOW fluidic system can be used to control flow rates, remove the air bubbles from the tubes, and change flow channels. In some instances, the ELVEFLOW fluidic system contains a flow sensor, pump, distributor, and flow controller.

[0135] B. Microscopy

[0136] For any of the systems described, the light source may be a light-emitting diode (LED) or one or more lasers (e.g., 1 laser, 2 lasers, 3 lasers, or more). In some instances, the light source is capable of emitting a light capable of inducing fluorescence of a fluorophore present on a primary labeling agent and / or a secondary labeling agent.

[0137] In some instances, the optical sensor includes a charge-coupled device (CCD), a photomultiplier tube (PMT), a photodiode, or a complementary metal-oxide-semiconductor (CMOS) device.

[0138] In some instances, the microscope, the light source, and the optical sensor are operatively coupled to the device to obtain one or more images e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, about 2000 images, about 3000 images, about 4000 images, about 5000 images, or more) from the channel.

[0139] A microscope setup that may be part of any of the systems described herein and / or be useful in any of the methods described herein may additionally include one or more lenses, mirrors (e.g., dichroic mirrors), collimators, condensers, objectives, etc. For example, a microscopy setup that may be part of any of the systems described herein and / or be useful in any of the methods described herein is a fluorescence microscope, e.g., a Zeiss upright automated epifluorescence microscope. In some instances, an exemplary microscopy setup may be part of any of the systems described herein and / or be useful in any of the methods described herein is shown in FIG. 8.

[0140] C. Computer

[0141] For any of the systems described, the computer may be operatively coupled to the fluidics control, the microscope, the light source, and / or the optical sensor. In some instances, the system’s computer may additionally be programmed to accept as input data one or more images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, 1000 images, or more) obtained using the system and provide as output data a determination that the target cells include cancer cells. In other instances, a computer may be programmed to accept as input data one or more images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, about 2000 images, about 3000 images, about 4000 images, about 5000 images, or more) obtained using any of the methods described and provide as output data a determination that the target cells includes cancer cells. In some instances, the input data contains one or more images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, about 2000 images, about 3000 images, about 4000 images, about 5000 images, or more) obtained using one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) primary and / or secondary labeling agents and one or more images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, about 2000 images, about 3000 images, about 4000 images, about 5000 images, or more) obtained using DAPI, in which the computer determines the position of target cells using the one or more DAPI images (e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, about 2000 images, about 3000 images, about 4000 images, about 5000 images, or more), and provides as output data a determination that the target cancer cells includes cancer cells using the one or more images(e.g., 1 image, 2 images, 3 images, 4 images, 5 images, 6 images, 7 images, 8 images, about 10 images, about 15 images, about 20 images, about 30 images, about 50 images, about 100 images, about 200 images, about 250 images, about 300 images, about 500 images, about 1000 images, about 2000 images, about 3000 images, about 4000 images, about 5000 images, or more) obtained using the one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more) primary and / or secondary labeling agents.

[0142] Embodiments

[0143] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

[0144] 1 . A microfluidic device comprising an inlet in fluid communication with a channel comprising an array comprising one or more rows of posts, wherein each row of posts comprises one or more capture areas and one or more passing areas, wherein the one or more capture areas comprises the posts and the passing area does not comprise any posts.

[0145] 2. The microfluidic device of paragraph 1 , wherein the microfluidic device comprises at least two rows of posts, wherein a first row of posts is adjacent to a second row of posts, and wherein:

[0146] (a) the geometric center of a first capture area in the first row does not align with the geometric center of any capture area in the second row along a direction substantially perpendicular to the rows; and / or

[0147] (b) the geometric center of a first passing area in the first row does not align with the geometric center of any passing area in the second row along a direction substantially perpendicular to the rows.

[0148] 3. The microfluidic device of paragraph 1 or 2, wherein each row comprises 1 to 50 capture areas and 1 to 50 passing areas.

[0149] 4. The microfluidic device of any one of paragraphs 1 -3, wherein each capture area comprises two to ten posts.

[0150] 5. The microfluidic device of any one of paragraphs 1 -4, wherein the microfluidic device comprises fifty to five hundred rows.

[0151] 6. The microfluidic device of any one of paragraphs 1 -5, wherein the rows are substantially parallel.

[0152] 7. The microfluidic device of any one of paragraphs 1 -6, wherein:

[0153] (a) each passing area is larger in size in a direction parallel to the row comprising the passing area than the distance between any two adjacent posts in a single capture area;

[0154] (b) the size of a passing area in a direction parallel to the row comprising the passing area of a first row of posts is different from the size of a passing area in a direction parallel to the row comprising the passing area of a second row of posts; and / or (c) the distance between any two adjacent posts in a single capture area of a first row of posts is different from the distance between any two adjacent posts in a single capture area of a second row of posts.

[0155] 8. The method of paragraph 7, wherein the size of a passing area in a direction substantially parallel to the row comprising the passing area is a first distance and the distance between any two adjacent posts in a single capture area is a second distance.

[0156] 9. A device comprising:

[0157] (a) an inlet; and

[0158] (b) a channel in fluidic communication with the inlet, wherein the channel comprises an array of posts positioned in one or more rows with a first distance and a second distance between adjacent posts in each row.

[0159] 10. The device of paragraph 8 or 9, wherein the first distance is at least 20 pm.

[0160] 11 . The device of paragraph 10, wherein the first distance is about 45 pm.

[0161] 12. The device of any one of paragraphs 8-11 , wherein the second distance is from about 15 pm to about 25 pm.

[0162] 13. The device of paragraph 12, wherein the second distance is about 22.5 pm.

[0163] 14. The device of any one of paragraphs 8-13, wherein each post:

[0164] (a) is T-shaped, L-shaped, triangular shaped, or semicircle shaped; and / or

[0165] (b) contains one or two protrusions.

[0166] 15. The device of paragraph 14, wherein each protrusion is from about 3 and about 8 pm.

[0167] 16. The device of paragraph 15, wherein each protrusion is about 5.25 pm.

[0168] 17. The device of any one of paragraphs 14-16, wherein the one or two protrusions of each post are substantially parallel to the row comprising the post.

[0169] 18. The device of any one of paragraphs 14-17, wherein the array of posts comprises a third distance between two adjacent protrusions of two adjacent posts of a row.

[0170] 19. The device of paragraph 18, wherein the third distance is from about 8 pm to about 14 pm.

[0171] 20. The device of paragraph 19, wherein the third distance is about 11 pm.

[0172] 21 . The device of any one of paragraphs 8-20, wherein the channel comprises a bottom surface, wherein the bottom surface comprises an optically transparent region.

[0173] 22. The device of paragraph 21 , wherein the optically transparent region comprises the array of posts.

[0174] 23. The device of any one of paragraphs 8-22, wherein the channel is fluidically sealed.

[0175] 24. The device of paragraph 23, wherein the channel comprises a top cover comprising an optically transparent cover region.

[0176] 25. The device of paragraph 24, wherein the optically transparent cover region comprises the array of posts.

[0177] 26. The device of paragraph 24 or 25, wherein the top cover comprises plastic, glass, or quartz.

[0178] 27. The device of any one of paragraphs 8-26, wherein each post comprises a height from about 25 pm to about 100 pm.

[0179] 28. The device of any one of paragraphs 8-27, wherein each post comprises a height of about 38 pm.

[0180] 29. The device of any one of paragraphs 8-28, wherein each post comprises a length of from about 12 pm to about 20 pm. 30. The device of paragraph 29, wherein each post comprises a length of about 14 pm.

[0181] 31 . The device of any one of paragraphs 8-30, wherein each row of the array of posts comprises one or more sets of six posts, wherein each set of six posts comprises two flanking posts (FP) and four inner posts (IP), arranged in the following configuration FP-I P-l P-l P-IP-FP in the row comprising the set of six posts.

[0182] 32. The device of paragraph 31 , wherein each FP has one protrusion and each IP has two protrusions.

[0183] 33. The device of paragraph 31 and 32, wherein:

[0184] (a) the distance between two adjacent FPs of two adjacent sets of six posts is the first distance;

[0185] (b) the distance between two adjacent posts of each set of six posts is the second distance; and / or

[0186] (c) the distance between two protrusions of two adjacent posts of each set of six posts is the third distance.

[0187] 34. The device of any one of paragraphs 8-33, wherein the channel is in fluidic communication with an outlet.

[0188] 35. The device of any one of paragraphs 8-34, wherein the inlet is in fluid communication with one or more reagent reservoirs.

[0189] 36. The device of paragraph 35, wherein the one or more reagent reservoirs comprise:

[0190] (a) a sample reservoir;

[0191] (b) a reagent reservoir; and / or

[0192] (c) a buffer reservoir.

[0193] 37. A method comprising:

[0194] (a) providing a device of any one of paragraphs 1 -36; and

[0195] (b) flowing a sample liquid from the inlet through the channel, wherein the sample liquid comprises a target cell.

[0196] 38. The method of paragraph 37, wherein:

[0197] (a) the target cell is smaller than the first distance and larger than the second distance; or

[0198] (b) the target cell is smaller than the first distance, smaller than the second distance, and larger than the third distance.

[0199] 39. The method of paragraph 37 or 38, further comprising flowing a first reagent liquid from the inlet through the channel, wherein the first reagent liquid comprises a primary labeling agent.

[0200] 40. The method of paragraph 39, wherein the primary labeling agent is capable of specific binding to the target cell.

[0201] 41 . The method of paragraph 39 or 40, wherein the primary labeling agent comprises a primary antibody or a small molecule, and wherein the primary labeling agent is capable of specific binding to a target on the target cell.

[0202] 42. The method of paragraph 41 , wherein the primary antibody comprises an anti-EpCAM antibody, an anti-EGFR antibody, an anti-MUC1 antibody, an anti-MARS1 antibody, an anti-HER2 antibody, anti- EpCAM antibody, an anti-ER antibody; an anti-PR antibody, an anti-Ki67 antibody, an anti-CD45 antibody, an anti-p53 antibody; an anti-ALK antibody, or a combination thereof.

[0203] 43. The method of paragraph 41 , wherein the small molecule comprises 4',6-diamidino-2-phenylindole (DAPI). 44. The method of any one of paragraphs 39-43, wherein the primary labeling agent comprises a detection moiety and / or a first nucleic acid.

[0204] 45. The method of paragraph 44, wherein the detection moiety is a fluorophore.

[0205] 46. The method of any one of paragraphs 39-45, further comprising flowing a second reagent liquid from the inlet through the channel, wherein the second reagent liquid comprises a secondary labeling agent.

[0206] 47. The method of paragraph 46, wherein the secondary labeling agent is capable of specific binding to the primary labeling agent.

[0207] 48. The method of paragraph 46 or 47, wherein the secondary labeling agent comprises a secondary antibody capable of binding to one or more of the primary antibodies and / or a second nucleic acid capable of hybridizing to the first nucleic acid.

[0208] 49. The method of any one of paragraphs 46-48, wherein the secondary labeling agent comprises a detection moiety.

[0209] 50. The method of paragraph 49, wherein the detection moiety is a fluorophore.

[0210] 51 . The method any one of paragraphs 37-50, further comprising flowing a wash buffer from the inlet through the channel.

[0211] 52. The method of paragraph 51 , wherein the wash buffer comprises a salt, a surfactant, bovine serum albumin, or a combination thereof.

[0212] 53. The method of paragraph 52, wherein the salt is monosodium phosphate, disodium phosphate, trisodium phosphate, or a combination thereof.

[0213] 54. The method of paragraph 53, wherein the surfactant is polysorbate 20.

[0214] 55. The method of any one of paragraphs 37-54, further comprising flowing an imaging buffer from the inlet through the channel.

[0215] 56. The method of any one of paragraphs 39-55, wherein the sample liquid, the first reagent liquid, the second reagent liquid, the wash buffer, and / or the imaging buffer are flown into the channel sequentially.

[0216] 57. The method of any one of paragraphs 37-56, wherein any of the sample liquid, the first reagent liquid, the second reagent liquid, and / or the wash buffer remain in the channel for a duration of time from about 5 seconds to about 30 minutes.

[0217] 58. The method of any one of paragraphs 39-57, wherein the flowing is at a flow rate of from about 5 pL / min to about 500 pL / min.

[0218] 59. The method of paragraph 58, wherein the flow rate is about 80 pL / min.

[0219] 60. The method of any one of paragraphs 39-58, wherein the flowing is for a duration from about 5 seconds to about 30 minutes.

[0220] 61 . The method of any one of paragraphs 37-60, wherein the sample liquid further comprises other particles.

[0221] 62. The method of paragraph 61 , wherein the other particles comprise cell debris, cell components, inorganic material, non-target cells, or a combination thereof.

[0222] 63. The method of paragraph 61 or 62, wherein non-target cells comprise blood cells, immune cells, fibrotic cells, or a combination thereof.

[0223] 64. The method of any one of paragraphs 61 -63, wherein the other particles are:

[0224] (a) smaller than the first distance and the second distance; or

[0225] (b) smaller than the first distance, the second distance, and the third distance. 65. The method of any one of paragraphs 37-64, further comprising obtaining one or more images of the channel.

[0226] 66. The method of paragraph 65, wherein the one or more images are obtained after flowing:

[0227] (a) the sample liquid and the first reagent liquid through the channel; or

[0228] (b) the sample liquid, the first reagent liquid, and the second reagent liquid through the channel.

[0229] 67. The method of paragraph 65 or 66, wherein the one or more images are obtained at a wavelength corresponding to one or more of the fluorophores on the primary labeling agent and / or the secondary labeling agent.

[0230] 68. The method of any one of paragraphs 65-67, further comprising analyzing the one or more images to identify the target cell.

[0231] 69. The method of any one of paragraphs 37-68, wherein the target cell is a cancer cell.

[0232] 70. The method of paragraph 69, wherein the target cell is an epithelial cell, a B cell, a T cell, or a glandular cell.

[0233] 71 . The method of any one of paragraphs 37-70, wherein the method is used to diagnose a disease.

[0234] 72. The method of paragraph 71 , wherein the disease is a cancer.

[0235] 73. The method of paragraph 72, wherein the cancer is:

[0236] (a) cholangiocarcinoma, pancreatic cancer, thyroid cancer, liver cancer, lung cancer, lymphoma, head and neck cancer, or breast cancer; and / or

[0237] (b) metastatic cancer.

[0238] 74. The method of any one of paragraphs 37-73, wherein the target cell was obtained from a subject using fine-needle aspiration (FNA), cytology brushing, swabbing, a blood sample, or a biopsy sample.

[0239] 75. A system suitable for use with the method of any one of paragraphs 37-74.

[0240] 76. A system comprising the device of any one of paragraphs 1 -36 operatively coupled with one or more of:

[0241] (a) a fluidics control;

[0242] (b) a microscope;

[0243] (c) a light source;

[0244] (d) an optical sensor;

[0245] (e) a waste container; and / or

[0246] (f) a computer.

[0247] 77. The system of paragraph 75 or 76, further comprising:

[0248] (a) a sample liquid comprising a target cell;

[0249] (b) a first reagent liquid comprising a primary labeling agent;

[0250] (c) a second reagent liquid comprising a secondary labeling agent;

[0251] (d) a wash buffer; and / or

[0252] (e) an imaging buffer.

[0253] 78. The system of paragraph 75 or 76, wherein the fluidics control comprises a pressure controller, a flow rate sensor, a flow switch controller, and / or a reagent splitter.

[0254] 79. The system of paragraph 78, wherein the pressure controller comprises a syringe pump, a peristaltic pump, a pressure-driven pump, or a micro-diaphragm pump. 80. The system of paragraph 78 or 79, wherein the fluidics control is operatively coupled with the device to flow a sample liquid, a first reagent liquid, a second reagent liquid, a wash buffer, and / or an imaging buffer into the channel.

[0255] 81 . The system of any one of paragraphs 76-80, wherein the light source comprises a light-emitting diode (LED) or one or more lasers.

[0256] 82. The system of paragraph 81 , wherein the light source is capable of emitting a light capable of inducing fluorescence of a fluorophore present on a primary labeling agent and / or a secondary labeling agent.

[0257] 83. The system of any one of paragraphs 76-82, wherein the optical sensor comprises a charge-coupled device (CCD), a photomultiplier tube (PMT), a photodiode, or a complementary metal-oxide- semiconductor (CMOS) device.

[0258] 84. The system of any one of paragraphs 76-83, wherein the microscope, the light source, and the optical sensor are operatively coupled to the device to obtain one or more images from the channel.

[0259] 85. The system of any one of paragraphs 76-84, wherein the computer is operatively coupled to the fluidics control, the microscope the light source, and / or the optical sensor.

[0260] 86. The system of any one of paragraphs 76-85, wherein the computer is programmed to accept as input data one or more images obtained using the system and provide as output data a determination that the target cells comprise cancer cells.

[0261] 87. A computer programmed to accept as input data one or more images obtained using the method of any one of paragraphs 37-74 and provide as output data a determination that the target cells comprise cancer cells.

[0262] 88. The computer of paragraph 87, the input data comprises one or more images obtained using one or more primary and / or secondary labeling agents and one or more images obtained using DAPI, wherein computer determines the position of target cells using the one or more DAPI images, and wherein the computer provides as output data a determination that the target cancer cells comprise cancer cells using the one or more images obtained using the one or more primary and / or secondary labeling agents.

[0263] Examples

[0264] Extreme cases of biliary tract cancer (cholangiocarcinoma) and metastasis were found in the lungs and brain. When suspicious cancer cells are found along the biliary tract, clinicians use a small brush to scrape cellular materials in the biliary wall. While the process is less invasive than putting a needle in a tissue, it results in even smaller amounts of cellular samples. Thus, the reported sensitivity of detecting cancer correctly is below 50%. In other words, when one examines 10 cancer patients, more than 5 patients are missed by chance. This is mainly due to the scarcity of collected samples in which (cyto)pathologists often say that there are enough samples to determine whether cancer is present.

[0265] In another example, when patients are diagnosed with cancer, clinicians often look at other organs to determine if the tumor spreads out of the originating organ (metastasis). Also, patients are monitored for recurrence after completing the cancer treatment. When clinicians find suspicious lesions in the brain (e.g., small lumps found in brain MRI / CT), performing a biopsy directly in the brain is clinically challenging. Alternatively, clinicians drain cerebrospinal fluids (CSF) and examine the presence of cancer cells, indicating that whether such suspicious lesions are likely cancer. Again, oftentimes, (cyto)pathologists cannot make the diagnosis due to insufficient samples.

[0266] The technology presented is specifically designed to address these current clinical unmet needs. The system was designed to be fast (within a couple of hours or less), accurate (by detecting cells with over-expression of specific tumor biomarkers), and compatible with current clinical work procedures (minimal disruption to clinicians). More importantly, a microfluidic chip was designed to enrich suspicious epithelial cells while disregarding irrelevant cells (e.g., blood cells) that could interfere with the examination as a background. The system thus includes 1 ) a small imaging flow cytometry system optimized for imaging and analyzing small numbers of cells, 2) a microfluidic chip that enriches target cells in the right field of view so that you don’t need to scan large areas to find your scarce samples, 3) microfluidic controllers with tubes filled with reagents (e.g., blocker, antibodies, etc.) for automatic operation with a minimal hand-on process, and 4) software to analyze the acquired images and determine if cancer cells are present. All these components need to operate fully in the clinical environment without disrupting the current workflow and increasing the clinical burden. With these functions, the system can now more accurately determine cancer than the current cytopathology examination.

[0267] The method differs from current cytopathology examination, which requires smearing, staining, and analyzing morphological features of cells to determine cancer cells. Lengthy manual processes are not needed, and the method uses molecular biomarkers instead of examining cell morphology. The molecular analysis provides new opportunities to identify drug-targetable key biomarkers (e.g., HER2 overexpression in breast cancer or EGFR in lung cancer). Currently, immunostaining and examination are barely done in these specimens because they require even more materials than the current cytopathology examination.

[0268] The method differs from molecular analysis of cells using a conventional flow cytometer, which requires a much larger number of cells (at least a few thousand) for reliable analysis.

[0269] This method furthers differs from other microfluidic technologies, as our system integrates multiple components (imaging, controller, analysis) to complete the analysis in a stand-alone device with minimal user requirements.

[0270] The following examples are provided as a description of how the methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0271] Table of Contents Example 1. Evaluation and validation of an iMAP system for capture and enrichment of cancer cells.

[0272] Objective

[0273] The objective of this study was to evaluate and validate the method of use for the iMAP system to reduce capture of debris and non-target cells from clinical samples and maximize cell capture efficiency.

[0274] Results

[0275] Cell Capture Efficiency and Immunolabelinq

[0276] The iMAP system’s cell capture efficiency and immunolabeling was first tested. After 10 min incubation of cells with primary antibodies, the cytology sample was injected into a microfluidic chip. Large suspicious epithelial cells were captured on the capture sites, while smaller blood cells, debris, and unbound antibodies were passed through. Then, fluorophore-conjugated secondary antibodies were flowed (FIG. 4). FIG. 4 shows 589 cells captured on the imaging site and labeled by secondary antibodies. A cocktail of antibodies was used against EpCAM, EGFR, and MUC1 (CCAsig) in the 475 nm channel and antibodies against MARS1 in the 630 nm channel. DAPI staining was done in the 385 nm channel. Comparing the captured cell counts before and after antibody labeling, there was no significant difference (P = 0.16, Welch’s t-test) with the average reduction rate of only 12.8% (FIG. 5-6), although the change was insignificant.

[0277] Next, the system’s ability to capture low numbers of cells was tested to ensure that the iMAP could capture and analyze rare cells in brush cytology samples. 12 - 50 SNU308 CCA cells were injected from serial dilution and counted the number of capture cells in the microfluidic chip. On average, a 91 .7% capture rate was achieved, as shown in FIG. 4, which shows that the iMAP can capture and analyze as few as 10 cells in a given sample. To test the quantitative accuracy of detecting marker-positive cells, SNU308 CCA cells were mixed with non-cancerous NIH / 3T3 cells in different ratios. After capturing the cells, the captured cells were immunolabeled using our CCAsig (a mixture of EpCAM, EGFR, and MUC1 ). The measured marker-positive cell counts showed a good linear correlation with the expected CCA cell counts (Pearson correlation coefficient r = 0.9981 , P = <0.0001 ; FIG.4). Collectively, the assay validation tests showed the iMAP’s ability to capture rare, suspicious cells and detect CCA cells using immunofluorescence staining and imaging.

[0278] The above-described results were obtained using the following materials and methods.

[0279] Materials and Methods

[0280] Fabrication and preparation for iMAP system

[0281] For the iMAP system, microfluidic chips were made of polydimethylsiloxane (PDMS) using a micro-patterned silicon wafer template. The micro-patterned silicon wafer was prepared by Heidelberg uPG501 (Heidelberg Instruments, Germany) using SU-8. Liquid silicone elastomer and curing agent with a weight ratio of 10:1 were thoroughly mixed. The PDMS mixture was degassed in a vacuum desiccator for 30 min. The PDMS mixture was slowly poured on the micro-patterned silicon wafer and then baked in the drying oven at 60 °C overnight to cure the PDMS. The cured PDMS was cut by a surgical knife along the pattern boundary, then peeled off from the silicon wafer and punched by a 2.5 mm biopsy puncher to make an inlet and outlet. The prepared PDMS and slide glass were treated with O2 plasma for 1 min for plasma bonding. Then, the treated side of each of them was attached and cured at 60 °C for 12 hours. The ELVEFLOW fluidic system was used to control flow rates, remove the air bubbles from the tubes, and change flow channels. The ELVEFLOW fluidic system includes a flow sensor, pump, distributor, and flow controller. With the fluidic system, the iMAP procedure was used for cell capturing and immunostaining.

[0282] Antibodies and Immunolabelinq

[0283] For immunostaining, antibodies were soluble in PBS mixed with 0.5% tween, 2.5 wt% of BSA for blocking, and 1 X BD Perm (BD Biosciences) buffer for permeabilization. Primary antibodies and secondary antibodies cocktail solutions were prepared. Cells were incubated in primary antibodies solution for 10 min and put in iMAP. The incubated cell solution was added to capture cells with an 80 pL / min flow rate for 2.75 min. Then, the PBST solution flowed with 0.4 bar for 15 sec and 80 pL / min for 4 min to remove unbound primary antibodies. The secondary antibody solution was injected with 30 pUmin for 5 min. Finally, the PBST solution flowed with 80 pUmin for 4 min to push out the excess secondary antibodies.

[0284] Image analysis

[0285] Fluorescence images of captured cells in the iMAPs were acquired using a Zeiss upright automated epifluorescence microscope. The acquired data were analyzed using CellProfiler software with a specific pipeline designed for this study. All cells were stained with DAPI, CCAsig, and MARS1 . During the image analysis process, DAPI-stained cells were first identified, and their outlines were delineated to define cell boundaries. Based on these identified DAPI-positive cells, fluorescence signal intensities were subsequently measured for CCAsig in the AF488 channel and MARS1 in the AF647 channel to evaluate their respective fluorescence

[0286] Example 2. Evaluation and validation of an iMAP system for specific immunolabeling and identification of cancer cells.

[0287] Objective

[0288] The objective of this study was to evaluate and validate the method of use for a microfluidic device described herein (e.g., the iMAP system) to maximize the signal-to-noise (S / N) of immunolabeling and optical system settings. Next, this improvement was validated to confirm the specificity of enrichment and immunolabeling for cancerous cell identification.

[0289] Results

[0290] Molecular characterization of CCA cells using iMAP

[0291] Candidate biomarkers CCAsig and MARS1 were tested. Those including CCAsig markers are well- characterized epithelial surface markers often over-expressed in solid cancer cells, including CCA. MARS1 is responsible for the methionylation of the initiator tRNA and showed over-expression in CCA tissues with immunohistochemistry. These biomarkers were characterized using CCA cell lines (SNU308, SNU478, SNU869) with a control fibroblast cell line (NIH / 3T3). FIG. 17 show NIH / 3T3 and SNU308 cells captured and stained in the iMAP system. The fluorescence images show single cells captured on the imaging site and positive for nuclear staining with DAPI. Immunofluorescence shows intense antibody staining of CCAsig markers on the cell membrane and MARS1 in the cytoplasm for SNU308 cells, while those markers were absent in NIH / 3T3 control cells.

[0292] The characterization was expanded for other CCA cell lines and their marker expressions were quantified (FIG. 17). For all CCA cell lines (SNU308, SNU478, SNU869) tested, the CCAsig markers and MARS1 showed elevated expression levels in CCA cell lines compared to NIH / 3T3 (FIG. 17, FIG. 19). The iMAP molecular characterization result was validated using the conventional flow cytometry analysis for the same cell lines (FIG. 22). The main difference is that conventional flow cytometry is unsuitable for analyzing scant cytology samples with low numbers of cells, while the iMAP system is specifically designed for the FNA sample analysis. The cut-off value was set to determine the marker positivity using signals from IgG isotype controls (FIG. 20). Based on the cut-off value, the marker-positive cell proportions were over 98-100% for CCAsig and 71 -90% for MARS1 , while 0% for NIH / 3T3 for both markers (FIG. 17). The result supports the use of the biomarkers for CCA cell detection. Similar validation was performed for the breast cancer cells (BT474, MCF7, MDAMB231 , SKBR3) (FIG. 21). For breast cancer, QUAD (EpCAM, EGFR, MUC1 , HER2) was used as breast cancer biomarkers, and HER2 and ER / PR were used for their molecular subtyping. The assay was tested and validated using four breast cancer cell lines, representing four different subtypes based on the positivity of HER2 and ER / PR, and a control cell line. The result shows good accuracies in detecting breast cancer cells using QUAD signals, HER2-positive cells (SKBR3 and BT474) using HER2 signals, and ER / PR-positive cells (MCF7 and BT474) using ER / PR signals. The system also showed good correlation using a conventional immunofluorescence imaging with a Zeiss microscope.

[0293] Immunolabelinq

[0294] In addition, The timeframe was evaluated for immunostaining that maximizes S / N of immunoblotted cells and determined that the optimal S / N for specific labeling occurred at around 10 minutes (FIG. 18).

[0295] Optical system and Graphical User Interface

[0296] Additional steps were taken to improve the optical system FIG. 7 - FIG. 13 and graphical user interface (FIG. 15). The fluidic chip design is outlined in FIG. 14. FIG. 7 shows an overview of a fluidic system and imaging system. (A) BT474 breast cancer cells were captured and imaged by an iMAP system, showing the multichannel imaging capability with one bright field (BF) and four fluorescence channels (DAPI at 405 nm, AF488 at 488 nm channel, AF555 at 555 nm channel, and AF647 at 647 nm channel). (B). The schematic illustration of an iMAP system, including an imaging system, a microfluidic chamber, a fluidic controller system, and a computer that connects the individual components and operates the system. FIG. 8 shows an exemplary iMAP optical system. The two photographs on the top show the fabricated imaging system. The CAD and optical schematic show how the optical system was designed in an epifluorescence configuration using a single multi-bandpass filter in front of a tube lens and multiple LEDs coupled with excitation filters. The graph and table on the right show the excitation and emission spectra of the filter sets. FIG. 9 shows an illumination simulation employing Zemax. The different lengths between condensing lens and LED (R1 ) and between focusing and condensing lenses (R2) were tested to identify the optimal distance lengths to achieve the maximum illumination power and uniformity. FIG. 10 shows an illumination simulation employing Zemax. Light sources with a single LED and four LEDs were compared. The single LED is located in the center of the light source while four LEDs were off-centered in a 2-by-2 configuration. The result shows the light source with four LEDs show 3-times bright light intensity power. FIG. 11 shows a comparison of CytoPAN2 and CytoPAN3 image quality. CytoPAN3 refers to an imaging system used in an iMAP system, while CytoPAN2 was a previously reported system (Adv Sci (Weinh). 2022;9(20):e2200415). The background signals and signal-to-noise ratios (SNRs) were compared between the two systems using microbeads coated with AF488 and AF647 fluorescence dyes. The results show the improvement of SNR in the CytoPAN3 approximately by 20-fold. FIG. 12 shows a comparison of image quality. Color (Sony IMX250LQR) and monochromatic CMOS camera (Sony IMX250LLR) sensors were compared for an iMAP imaging system. Both cameras used the same Sony IMX250 image sensor. The result shows the color CMOS showed better SNR in green and red channels compared to the monochromatic camera, likely due to lower background noise with an RGB filter implemented in the color CMOS camera. FIG. 13 shows spectral unmixing. (A) When using a multibandpass filter with multiple excitation LED light sources, fluorescence signals often bleed through adjacent channels. (B) This bleed-through can be corrected by unmixing the fluorescence signals by calculating the ratio of bleed-through for each dye to other channels. (C) The matrix calculation, showing the unmixing method. The system matrix (S, 12 x 5) multiplied by the true signals (T, 5x1 ) is recorded in the image camera (C, 12x1 ). From the unmixing calculation, the true signals can be calculated by T = C X s-1.

[0297] FIG. 14 shows an exemplary fluidic chip design. The left shows the mask design to fabricate 24 fluidic chips on a 4” silicon wafer. The middle shows a single fluidic chip design. The right shows a zoomed-in image in the area of cell capture.

[0298] FIG. 15 shows an exemplary graphical user interface. The left is a microscope GUI that controls an imaging system of iMAP. The right is a fluidics GUI that controls fluidic sequences, flow rates, and time duration.

[0299] The above-described results were obtained using the following materials and methods.

[0300] Materials and Methods Cell lines

[0301] A panel of CCA cell lines with different expressions of CCAsig (EpCAM, EGFR, MUC1 ) and MARS1 markers was used for validation: NIH / 3T3, SNU308, SNU478, and SNU869. NIH / 3T3 was purchased from ATCC and grown in Dulbecco’s minimum essential medium (DMEM) (Hyclone) with 10% fetal bovine serum (FBS) (Gibco, Invitrogen). SNU308, SNU478, and SNU869 were provided by Yonsei University and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen) supplemented with 10% FBS.

[0302] Flow cytometry After trypsinizing NIH / 3T3, H69, SNU308, SNU478, and SNU869 cells, they were washed with PBS and fixed with CytoRich Red (Thermo Scientific, MA, USA) to keep them in 4 °C. The fixed cells were blocked with 0.5% BSA in PBS for 30 min and incubated with primary antibodies for 1 h. The AF488 anti-mouse secondary antibodies were used to bind the primary antibodies for 30 min. CytoFlex flow cytometer (Beckman Coulter) using the 488 / 8 nm bandpass filter for FSC (48 V), SSC (29 V), and FITC (20 V). The data were analzed with FlowJo X 10.0 with median fluorescence intensity. The isotype control was used for the normalization of each cell.

[0303] Image analysis

[0304] Fluorescence images of captured cells in the iMAPs were acquired using a Zeiss upright automated epifluorescence microscope. The acquired data were analyzed using Cell Profiler software with a specific pipeline designed for this study. All cells were stained with DAPI, CCAsig, and MARS1 . During the image analysis process, DAPI-stained cells were first identified, and their outlines were delineated to define cell boundaries. Based on these identified DAPI-positive cells, fluorescence signal intensities were subsequently measured for CCAsig in the AF488 channel and MARS1 in the AF647 channel to evaluate their respective fluorescence intensities. The signal intensity was then used to determine positivity by applying a cutoff value, which was defined as the mean intensity of IgG controls ± 2 x standard deviation.

[0305] Example 3. Use of iMAP system for rapid CCA diagnosis.

[0306] This example describes a method to use the iMAP system with brushing cytology clinical samples for cholangiocarcinoma (CCA) diagnosis.

[0307] Objective

[0308] The objective of this study was to evaluate the usage of the integrated multi-channel automatic profiling (iMAP) system for Cholangiocarcinoma (CCA) diagnosis from brushing cytology samples. This study determined the optimal conditions to distinguish between CCA cancer cells and benign cells. In a pilot clinical study involving 37 patients, good clinical diagnostic accuracy (AUC = 0.82) was demonstrated for CCA using a four-marker combination (MUC1 , EpCAM, EGFR, and MARS1 ). In addition, this study evaluated the differences in CCA biomarkers expression to distinguish cancerous and benign cells when used with the iMAP.

[0309] Results

[0310] Design and analysis procedure of iMAP

[0311] Brush cytology specimens from ERCP contain a small number of cancer cells mixed with blood cells, immune cells, fibrotic cells, and others. The system was designed to analyze unprocessed clinical specimens. In our system, CCA cells from clinical brush cytology samples were automatically captured, washed, stained, and analyzed for molecular markers, all in a single chip (FIG. 2). First, a collected ERCP sample was inserted in a tube containing fixatives and blocking solutions. The sample was then introduced to a microfluidic chip with varying sizes of capture sites to i) filter out large cell aggregates, ii) capture suspicious large (> 11 pm) epithelial cells, and iii) pass through smaller non-tumor cells. This capturing was done by the size differential between epithelial cells and other blood cells, which minimizes sample loss while enriching suspicious cells captured in an imaging area for molecular profiling. The cell capture area in 18 pm x 14 pm x 38 pm has a bottleneck-like structure with 22.5 pm of wide inlet and 11 pm of narrow outlet (FIG. 5). Large epithelial cells were captured in the designed area with a flow through the capture site. A wider gap of 45 pm was used between capture sites to allow a continuous flow of cells and antibody / washing solutions after the capture sites are fully occupied. To further simplify the assay procedure and time, the collected clinical specimen was directly added to primary antibodies for immunostaining. The sample injection and cell capture were done in 3 min, followed by 4 min washing with PBST solution. After cell capture, secondary antibodies were introduced to fluorescently label the captured cells via primary antibody staining for 5 min, followed by washing for 4 min. A microfluidic flow controller (OB1 , ELVEFLOW) was used to flow different solutions automatically. The captured cells were then imaged by a fluorescence microscope with a 10x objective lens (NA = 0.25). Software was used to analyze the fluorescence images to quantify captured cell counts and marker intensities in individual cells. The overall process, from sample injection to data analysis, took 30 min. FIG. 23 shows CCA diagnosis analysis for (A) Heatmap of all patient samples from each marker including, CCAsig and MARS1 (the color scale indicates the number of positive cells). Comparison for (B) the number of total captured cells, (C) the number of CCAsig positive cells, (D) the number of MARS1 positive cells, and (E) the number of CCAsig or MARS1 positive cells between CCA and benign (Mann-Whitney t-test). (F) A waterfall plot of CCA and benign for the number of CCAsig or MARS1 positive cells. (G) Receiver operating characteristic (ROC) curves of CCAsig-positive (AUC = 0.80), MARS1 -positive cells (AUC = 0.74), and CCAsig or MARS1 -positive cells (AUC = 0.82).

[0312] Conclusion

[0313] CCA is a challenging cancer to diagnose, necessitating new diagnostic methods to improve the current tissue-based diagnosis. The imminent challenge is that a significant portion of cytology samples are non-diagnostic due to the paucity of sampled cells. As illustrated in FIG. 2, the iMAP system specifically addresses the problems of low cell counts, automated sample processing and multiplexed analyses. As shown in FIG. 4 - FIG. 6, target epithelial cells were enriched for molecular diagnosis by leveraging a microfluidic chip with immunofluorescence staining of the target suspicious cells. The pilot clinical test demonstrates this approach for clinical applications, as shown in. Assay conditions and reagent concentrations were optimized to simplify the assay process to be completed within one hour, as shown by FIG. 16 and FIG. 18. The microfluidic chip facilitates the enrichment of target cells within the imaging area, and a pre-programmed fluidic controller sequentially flows various solutions for immunostaining and washing. The iMAP system exhibits excellent correlation with the conventional flow cytometry system for molecular analysis, as shown by FIG. 22. Nevertheless, it still necessitates a significantly smaller number of cells, typically obtained through brushing biopsy of the biliary tract.

[0314] The above-described results were obtained using the following materials and methods.

[0315] Materials and methods

[0316] The method is diagrammed in FIG. 2 and summarized below.

[0317] Cytology samples are collected from patients with suspected CCA using bile duct brushing and loaded into a processing solution containing cancer-specific markers (CCAsig and MARS1 ). Next, the sample is introduced into an automated microfluidic immunoassay system, where a fluidic control system regulates the flow of reagents. The microfluidic chip captures target cancer cells, immunostains them with specific antibodies, and performs washing steps to remove non-specific debris. These immunostained cells are rapidly imaged using fluorescence microscopy to detect cancer biomarkers. Then, the captured fluorescence images are analyzed using CellProfiler software, where computational processing enables automated identification and classification of target cells. Quantitative data, including biomarker intensity and cell counting ratios, are used for diagnosis. The system determines the percentage of normal versus cancerous cells, providing a diagnostic result (positive or negative) within 30 minutes.

[0318] Cell lines

[0319] A panel of CCA cell lines with different expressions of CCAsig (EpCAM, EGFR, MUC1 ) and MARS1 markers was used for validation: NIH / 3T3, SNU308, SNU478, and SNU869. NIH / 3T3 was purchased from ATCC and grown in Dulbecco’s minimum essential medium (DMEM) (Hyclone) with 10% fetal bovine serum (FBS) (Gibco, Invitrogen). SNU308, SNU478, and SNU869 were provided by Yonsei University and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen) supplemented with 10% FBS.

[0320] Fabrication and preparation for iMAP

[0321] For the iMAP system, microfluidic chips were made of polydimethylsiloxane (PDMS) using a micro-patterned silicon wafer template. The micro-patterned silicon wafer was prepared by Heidelberg uPG501 (Heidelberg Instruments, Germany) using SU-8. Liquid silicone elastomer and curing agent with a weight ratio of 10:1 were thoroughly mixed. The PDMS mixture was degassed in a vacuum desiccator for 30 min. The PDMS mixture was slowly poured on the micro-patterned silicon wafer and then baked in the drying oven at 60 °C overnight to cure the PDMS. The cured PDMS was cut by a surgical knife along the pattern boundary, then peeled off from the silicon wafer and punched by a 2.5 mm biopsy puncher to make an inlet and outlet. The prepared PDMS and slide glass were treated with O2 plasma for 1 min for plasma bonding. Then, the treated side of each of them was attached and cured at 60 °C for 12 hours. The ELVEFLOW fluidic system was used to control flow rates, remove the air bubbles from the tubes, and change flow channels. The ELVEFLOW fluidic system includes a flow sensor, pump, distributor, and flow controller. With the fluidic system, the iMAP procedure was used for cell capturing and immunostaining.

[0322] Antibodies and immunostaininq

[0323] Table 1 : List of antibodies and dilution factors used in iMAP.

[0324] Table 1 lists the antibodies and dilutions used in this research. For immunostaining, antibodies were soluble in PBS mixed with 0.5% tween, 2.5 wt% of BSA for blocking, and 1X BD Perm buffer (BD Biosciences) for permeabilization. Primary antibodies and secondary antibodies cocktail solutions were prepared. Cells were incubated in primary antibodies solution for 10 min and put in iMAP. The incubated cell solution was added to capture cells with an 80 pL / min flow rate for 2.75 min. Then, the PBST solution flowed with 0.4 bar for 15 sec and 80 pL / min for 4 min to remove unbound primary antibodies. The secondary antibody solution was injected with 30 pL / min for 5 min. Finally, the PBST solution flowed with 80 pL / min for 4 min to push out the excess secondary antibodies (Table 1).

[0325] Image analysis

[0326] Fluorescence images of captured cells in the iMAPs were acquired using a Zeiss upright automated epifluorescence microscope. The acquired data were analyzed using CellProfiler software with a specific pipeline designed for this study. All cells were stained with DAPI, CCA, and MARS1 . During the image analysis process, DAPI-stained cells were first identified, and their outlines were delineated to define cell boundaries. Based on these identified DAPI-positive cells, fluorescence signal intensities were subsequently measured for CCA in the AF488 channel and MARS1 in the AF647 channel to evaluate their respective fluorescence intensities. The signal intensity was then used to determine positivity by applying a cutoff value, which was defined as the mean intensity of IgG controls ± 2 x standard deviation.

[0327] Flow cytometry

[0328] After trypsinizing NIH / 3T3, H69, SNU308, SNU478, and SNU869 cells, they were washed with PBS and fixed with CytoRich Red (Thermo Scientific, MA, USA) to keep them in 4°C. The fixed cells were blocked with 0.5% BSA in PBS for 30 min and incubated with primary antibodies for 1 h. The AF488 anti- mouse secondary antibodies were used to bind the primary antibodies for 30 min. CytoFlex flow cytometer (Beckman Coulter) using the 488 / 8 nm bandpass filter for FSC (48 V), SSC (29 V), and FITC (20 V). The data were analyzed with FlowJo X 10.0 with median fluorescence intensity. The isotype control was used for the normalization of each cell.

[0329] Clinical study

[0330] Bile duct brush samples of patients with CCA or benign strictures were maintained by 5-8 passing across the lesion during ERCP using a GRBH-230-3-3.5 brush (Wilson-Cook Medical, Inc, Winston- Salem, NC, USA). The brushed samples were subsequently washed in RPMI-1640 medium (Gibco BRL, Rockwell, Md, USA) and produced using ThinPrep (Cytyc Co, Marlborough, Mass, USA). The frozen samples were thawed and centrifuged with 300 g 4 °C for 3 min. The supernatant was removed, and the tissue pellet was washed with PBS 500 pL. The samples were centrifuged with 300 g 4 °C for 3 min once again and diluted with 100-200 pL depending on the tissue volume. Table 2 states the clinical characteristics of patients diagnosed with CCA and with benign samples used in this example.

[0331] Table 2: Clinical characteristics of patients diagnosed with CCA and benign.

[0332] . .. Cholangiocarc noma > .

[0333] Characteristics Benign

[0334] (CCA)

[0335] Total cases 25 12

[0336] Age (years)

[0337] Median 69 72

[0338] Range 51-92 53-84

[0339] Sex

[0340] Male 16 6

[0341] Female 9 6

[0342] Example 4. Use of iMAP system for rapid breast cancer diagnosis.

[0343] This example describes a method to use the iMAP system with clinical samples including fine needle aspiration (FNA), cerebrospinal fluid (CSF), or pericardial Effusion (PE) for breast cancer diagnosis.

[0344] Objective

[0345] The objective of this study was to evaluation the usage of the integrated multi-channel automatic profiling (iMAP) system for breast cancer diagnosis from clinical samples including fine needle aspiration (FNA), cerebrospinal fluid (CSF), or pericardial Effusion (PE). This study determined the optimal conditions to distinguish between cancer cells and benign cells. In addition, this study evaluated the differences in biomarkers for cells procured from FNA, CSF and PE sample types. Results

[0346] This study found that previously established biomarkers for breast cancer (HER2, Quad, ER / PR) were useful in distinguishing malignant breast cells from benign cells with the iMAP.

[0347] Fine Needle Aspirate (FNA) Clinical Samples

[0348] FIG. 24 shows that Quad is differentially labeled on benign and malignant cells found in FNA samples. ER and PR labeling were not significant. FIG. 25 shows that diagnostic sensitivity and selectivity with Quad marker, as shown by AUC, improved when multiple FNA samples from the same patient were used.

[0349] Pericardial Effusion (PE) Clinical Samples

[0350] FIG. 26 shows that quad is a biomarker that is differentially labeled on benign and malignant cells found in PE samples for suspected metastasis of breast cancer patients in lung.

[0351] Cerebrospinal Fluid (CSF) Clinical Samples

[0352] FIG. 27 shows that quad is differentially labeled on benign and malignant cells found in CSF samples for suspected metastasis of breast cancer patients in brain.

[0353] Clinical markers (CSF, FNA, PE)

[0354] FIG. 28 shows that regardless of which clinical sample type was used FNA, PE, or CSF, the quad marker demonstrated high diagnostic performance. For clinical samples overall, a statistically significant difference was observed, and ROC analysis showed a high AUC of 0.96.

[0355] Conclusion

[0356] As shown in FIG. 1 , Usage of iMAP system for rapid breast cancer diagnosis requires less sample to enable diagnosis and results in reduced variability between individuals compared to alternative methods such as FACS. FACS requires a large amount of samples, at least a few thousand cells, for reliable analysis of marker expressions in cells, which is inadequate in processing scant cytology samples. In addition, as shown in FIG. 3, the entire diagnostic workflow takes less than 30 minutes.

[0357] The above-described results were obtained using the following materials and methods.

[0358] Materials and methods

[0359] The method is diagrammed in FIG. 1 and summarized below.

[0360] FNA, PE, CSF Clinical samples are collected from patients with suspected breast and loaded into a processing solution containing cancer-specific markers (Quad, ER / PR, HER2). Next, the sample is introduced into an automated microfluidic immunoassay system, where a fluidic control system regulates the flow of reagents. The microfluidic chip captures target cancer cells, immunostains them with specific antibodies, and performs washing steps to remove non-specific debris. These immunostained cells are rapidly imaged using fluorescence microscopy to detect cancer biomarkers. Then, the captured fluorescence images are analyzed using CellProfiler software, where computational processing enables automated identification and classification of target cells. Quantitative data, including biomarker intensity and cell counting ratios, are used for diagnosis. The system determines the percentage of normal versus cancerous cells, providing a diagnostic result (positive or negative) within 30 minutes.

[0361] Cell lines

[0362] A panel of breast cancer cell lines representing four different molecular subtypes based on HER2 and ER / PR expressions was used for validation: MDAMB231 , MCF7, SKBR3, BT474. Jurkats, which are not breast cancer cells, were used as a negative control for marker and method validation according to standard methods.

[0363] Fabrication and preparation for iMAP

[0364] For the iMAP system, microfluidic chips were made of polydimethylsiloxane (PDMS) using a micro-patterned silicon wafer template. The micro-patterned silicon wafer was prepared by Heidelberg uPG501 (Heidelberg Instruments, Germany) using SU-8. Liquid silicone elastomer and curing agent with a weight ratio of 10:1 were thoroughly mixed. The PDMS mixture was degassed in a vacuum desiccator for 30 min. The PDMS mixture was slowly poured on the micro-patterned silicon wafer and then baked in the drying oven at 60 °C overnight to cure the PDMS. The cured PDMS was cut by a surgical knife along the pattern boundary, then peeled off from the silicon wafer and punched by a 2.5 mm biopsy puncher to make an inlet and outlet. The prepared PDMS and slide glass were treated with O2 plasma for 1 min for plasma bonding. Then, the treated side of each of them was attached and cured at 60 °C for 12 hours. The ELVEFLOW fluidic system was used to control flow rates, remove the air bubbles from the tubes, and change flow channels. The ELVEFLOW fluidic system includes a flow sensor, pump, distributor, and flow controller. With the fluidic system, the iMAP procedure was used for cell capturing and immunostaining.

[0365] Image analysis

[0366] Fluorescence images of captured cells in the iMAPs were acquired using a Zeiss upright automated epifluorescence microscope. The acquired data were analyzed using CellProfiler software with a specific pipeline designed for this study. All cells were stained with DAPI, QUAD in AF488, HER2 in AF555, and ER / PR in AF647 (see Table 3).

[0367] Table 3: Primary and Secondary Antibodies Used

[0368] During the image analysis process, DAPI-stained cells were first identified, and their outlines were delineated to define cell boundaries. Based on these identified DAPI-positive cells, fluorescence signal intensities were subsequently measured for in the Alexa Fluor 488 (AF488) channel and in the Alexa Fluor 647 (AF647) channel to evaluate their respective fluorescence - intensities. The signal intensity was then used to determine positivity by applying a cutoff value, which was defined as the mean intensity of IgG controls ± 2 x standard deviation.

[0369] Flow cytometry

[0370] After trypsinizing cells, they were washed with PBS and fixed with CytoRich Red (Thermo Scientific, MA, USA) to keep them in 4 °C. The fixed cells were blocked with 0.5% BSA in PBS for 30 min and incubated with primary antibodies for 1 h. The AF488 anti-mouse secondary antibodies were used to bind the primary antibodies for 30 min. CytoFlex flow cytometer (Beckman Coulter) using the 488 / 8 nm bandpass filter for FSC (48 V), SSC (29 V), and FITC (20 V). Data were analyzed with FlowJo X 10.0 with median fluorescence intensity. The isotype control was used for the normalization of each cell.

[0371] Clinical study

[0372] Clinical samples were obtained from breast cancer or benign patients. For FNA, fine-needle aspiration was performed for surgically removed tissue samples from patients with known pathological diagnoses. For PE and CSF, the bio fluids were drained through routine clinical procedures with suspected metastasis of breast cancer in lung and brain, respectively.

[0373] Other Embodiments

[0374] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0375] Other embodiments are within the claims.

[0376] What is claimed is:

Claims

Claims1 . A microfluidic device comprising an inlet in fluid communication with a channel comprising an array comprising one or more rows of posts, wherein each row of posts comprises one or more capture areas and one or more passing areas, wherein the one or more capture areas comprises the posts and the passing area does not comprise any posts.

2. The microfluidic device of claim 1 , wherein the microfluidic device comprises at least two rows of posts, wherein a first row of posts is adjacent to a second row of posts, and wherein:(a) the geometric center of a first capture area in the first row does not align with the geometric center of any capture area in the second row along a direction substantially perpendicular to the rows; and / or(b) the geometric center of a first passing area in the first row does not align with the geometric center of any passing area in the second row along a direction substantially perpendicular to the rows.

3. The microfluidic device of claim 1 , wherein each row comprises 1 to 50 capture areas and 1 to 50 passing areas.

4. The microfluidic device of claim 1 , wherein each capture area comprises two to ten posts.

5. The microfluidic device of claim 1 , wherein the microfluidic device comprises fifty to five hundred rows.

6. The microfluidic device of claim 1 , wherein the rows are substantially parallel.

7. The microfluidic device of claim 1 , wherein:(a) each passing area is larger in size in a direction parallel to the row comprising the passing area than the distance between any two adjacent posts in a single capture area;(b) the size of a passing area in a direction parallel to the row comprising the passing area of a first row of posts is different from the size of a passing area in a direction parallel to the row comprising the passing area of a second row of posts; and / or(c) the distance between any two adjacent posts in a single capture area of a first row of posts is different from the distance between any two adjacent posts in a single capture area of a second row of posts.

8. The microfluidic device of claim 7, wherein the size of a passing area in a direction substantially parallel to the row comprising the passing area is a first distance and the distance between any two adjacent posts in a single capture area is a second distance.

9. A device comprising:(a) an inlet; and(b) a channel in fluidic communication with the inlet, wherein the channel comprises an array of posts positioned in one or more rows with a first distance and a second distance between adjacent posts in each row.

10. The device of claim 8 or 9, wherein the first distance is at least 20 pm.1 1 . The device of claim 10, wherein the first distance is about 45 pm.

12. The device of claim 8 or 9, wherein the second distance is from about 15 pm to about 25 pm.

13. The device of claim 12, wherein the second distance is about 22.5 pm.

14. The device of claim 8 or 9, wherein each post:(a) is T-shaped, L-shaped, triangular shaped, or semicircle shaped; and / or(b) contains one or two protrusions.

15. The device of claim 14, wherein each protrusion is from about 3 and about 8 pm.

16. The device of claim 15, wherein each protrusion is about 5.25 pm.

17. The device of claim 14, wherein the one or two protrusions of each post are substantially parallel to the row comprising the post.

18. The device of claim 14, wherein the array of posts comprises a third distance between two adjacent protrusions of two adjacent posts of a row.

19. The device of claim 18, wherein the third distance is from about 8 pm to about 14 pm.

20. The device of claim 19, wherein the third distance is about 1 1 pm.21 . The device of claim 8 or 9, wherein the channel comprises a bottom surface, wherein the bottom surface comprises an optically transparent region.

22. The device of claim 21 , wherein the optically transparent region comprises the array of posts.

23. The device of claim 8 or 9, wherein the channel is fluidically sealed.

24. The device of claim 23, wherein the channel comprises a top cover comprising an optically transparent cover region.

25. The device of claim 24, wherein the optically transparent cover region comprises the array of posts.

26. The device of claim 24, wherein the top cover comprises plastic, glass, or quartz.

27. The device of claim 8 or 9, wherein each post comprises a height from about 25 pm to about 100 pm.

28. The device of claim 8 or 9, wherein each post comprises a height of about 38 pm.

29. The device of claim 8 or 9, wherein each post comprises a length of from about 12 pm to about 20 pm.

30. The device of claim 29, wherein each post comprises a length of about 14 pm.31 . The device of claim 8 or 9, wherein each row of the array of posts comprises one or more sets of six posts, wherein each set of six posts comprises two flanking posts (FP) and four inner posts (IP), arranged in the following configuration FP-IP-I P-l P-l P-FP in the row comprising the set of six posts.

32. The device of claim 31 , wherein each FP has one protrusion and each IP has two protrusions.

33. The device of claim 31 , wherein:(a) the distance between two adjacent FPs of two adjacent sets of six posts is the first distance;(b) the distance between two adjacent posts of each set of six posts is the second distance; and / or(c) the distance between two protrusions of two adjacent posts of each set of six posts is the third distance.

34. The device of claim 8 or 9, wherein the channel is in fluidic communication with an outlet.

35. The device of claim 8 or 9, wherein the inlet is in fluid communication with one or more reagent reservoirs.

36. The device of claim 35, wherein the one or more reagent reservoirs comprise:(a) a sample reservoir;(b) a reagent reservoir; and / or(c) a buffer reservoir.

37. A method comprising:(a) providing a device of claim 8 or 9; and(b) flowing a sample liquid from the inlet through the channel, wherein the sample liquid comprises a target cell.

38. The method of claim 37, wherein:(a) the target cell is smaller than the first distance and larger than the second distance; or(b) the target cell is smaller than the first distance, smaller than the second distance, and larger than the third distance.

39. The method of claim 37, further comprising flowing a first reagent liquid from the inlet through the channel, wherein the first reagent liquid comprises a primary labeling agent.

40. The method of claim 39, wherein the primary labeling agent is capable of specific binding to the target cell.41 . The method of claim 39, wherein the primary labeling agent comprises a primary antibody or a small molecule, and wherein the primary labeling agent is capable of specific binding to a target on the target cell.

42. The method of claim 41 , wherein the primary antibody comprises an anti-EpCAM antibody, an anti- EGFR antibody, an anti-MUC1 antibody, an anti-MARS1 antibody, an anti-HER2 antibody, anti-EpCAM antibody, an anti-ER antibody; an anti-PR antibody, an anti-Ki67 antibody, an anti-CD45 antibody, an anti-p53 antibody; an anti-ALK antibody, or a combination thereof.

43. The method of claim 41 , wherein the small molecule comprises 4',6-diamidino-2-phenylindole (DAPI).

44. The method of claim 39, wherein the primary labeling agent comprises a detection moiety and / or a first nucleic acid.

45. The method of claim 44, wherein the detection moiety is a fluorophore.

46. The method of claim 39, further comprising flowing a second reagent liquid from the inlet through the channel, wherein the second reagent liquid comprises a secondary labeling agent.

47. The method of claim 46, wherein the secondary labeling agent is capable of specific binding to the primary labeling agent.

48. The method of claim 46, wherein the secondary labeling agent comprises a secondary antibody capable of binding to one or more of the primary antibodies and / or a second nucleic acid capable of hybridizing to the first nucleic acid.

49. The method of claim 46, wherein the secondary labeling agent comprises a detection moiety.

50. The method of claim 49, wherein the detection moiety is a fluorophore.51 . The method claim 37, further comprising flowing a wash buffer from the inlet through the channel.

52. The method of claim 51 , wherein the wash buffer comprises a salt, a surfactant, bovine serum albumin, or a combination thereof.

53. The method of claim 52, wherein the salt is monosodium phosphate, disodium phosphate, trisodium phosphate, or a combination thereof.

54. The method of claim 53, wherein the surfactant is polysorbate 20.

55. The method of claim 37, further comprising flowing an imaging buffer from the inlet through the channel.

56. The method of claim 39, wherein the sample liquid, the first reagent liquid, the second reagent liquid, the wash buffer, and / or the imaging buffer are flown into the channel sequentially.

57. The method of claim 37, wherein any of the sample liquid, the first reagent liquid, the second reagent liquid, and / or the wash buffer remain in the channel for a duration of time from about 5 seconds to about 30 minutes.

58. The method of claim 39, wherein the flowing is at a flow rate of from about 5 pL / min to about 500 pL / min.

59. The method of claim 58, wherein the flow rate is about 80 pL / min.

60. The method of claim 39, wherein the flowing is for a duration from about 5 seconds to about 30 minutes.61 . The method of claim 37, wherein the sample liquid further comprises other particles.

62. The method of claim 61 , wherein the other particles comprise cell debris, cell components, inorganic material, non-target cells, or a combination thereof.

63. The method of claim 62, wherein non-target cells comprise blood cells, immune cells, fibrotic cells, or a combination thereof.

64. The method of claim 61 , wherein the other particles are:(a) smaller than the first distance and the second distance; or(b) smaller than the first distance, the second distance, and the third distance.

65. The method of claim 37, further comprising obtaining one or more images of the channel.

66. The method of claim 65, wherein the one or more images are obtained after flowing:(a) the sample liquid and the first reagent liquid through the channel; or(b) the sample liquid, the first reagent liquid, and the second reagent liquid through the channel.

67. The method of claim 65, wherein the one or more images are obtained at a wavelength corresponding to one or more of the fluorophores on the primary labeling agent and / or the secondary labeling agent.

68. The method of claim 65, further comprising analyzing the one or more images to identify the target cell.

69. The method of claim 37, wherein the target cell is a cancer cell.

70. The method of claim 69, wherein the target cell is an epithelial cell, a B cell, a T cell, or a glandular cell.71 . The method of claim 37, wherein the method is used to diagnose a disease.

72. The method of claim 71 , wherein the disease is a cancer.

73. The method of claim 72, wherein the cancer is:(a) cholangiocarcinoma, pancreatic cancer, thyroid cancer, liver cancer, lung cancer, lymphoma, head and neck cancer, or breast cancer; and / or(b) metastatic cancer.

74. The method of claim 37, wherein the target cell was obtained from a subject using fine-needle aspiration (FNA), cytology brushing, swabbing, a blood sample, or a biopsy sample.

75. A system suitable for use with the method of claim 37.

76. A system comprising the device of claim 8 or 9 operatively coupled with one or more of:(a) a fluidics control;(b) a microscope;(c) a light source;(d) an optical sensor;(e) a waste container; and / or(f) a computer.

77. The system of claim 76, further comprising:(a) a sample liquid comprising a target cell;(b) a first reagent liquid comprising a primary labeling agent;(c) a second reagent liquid comprising a secondary labeling agent;(d) a wash buffer; and / or(e) an imaging buffer.

78. The system of claim 76, wherein the fluidics control comprises a pressure controller, a flow rate sensor, a flow switch controller, and / or a reagent splitter.

79. The system of claim 78, wherein the pressure controller comprises a syringe pump, a peristaltic pump, a pressure-driven pump, or a micro-diaphragm pump.

80. The system of claim 78, wherein the fluidics control is operatively coupled with the device to flow a sample liquid, a first reagent liquid, a second reagent liquid, a wash buffer, and / or an imaging buffer into the channel.81 . The system of claim 76, wherein the light source comprises a light-emitting diode (LED) or one or more lasers.

82. The system of claim 81 , wherein the light source is capable of emitting a light capable of inducing fluorescence of a fluorophore present on a primary labeling agent and / or a secondary labeling agent.

83. The system of claim 76, wherein the optical sensor comprises a charge-coupled device (CCD), a photomultiplier tube (PMT), a photodiode, or a complementary metal-oxide-semiconductor (CMOS) device.

84. The system of claim 76, wherein the microscope, the light source, and the optical sensor are operatively coupled to the device to obtain one or more images from the channel.

85. The system of claim 76, wherein the computer is operatively coupled to the fluidics control, the microscope the light source, and / or the optical sensor.

86. The system of claim 76, wherein the computer is programmed to accept as input data one or more images obtained using the system and provide as output data a determination that the target cells comprise cancer cells.

87. A computer programmed to accept as input data one or more images obtained using the method of claim 37 and provide as output data a determination that the target cells comprise cancer cells.

88. The computer of claim 87, the input data comprises one or more images obtained using one or more primary and / or secondary labeling agents and one or more images obtained using DAPI, wherein computer determines the position of target cells using the one or more DAPI images, and wherein the computer provides as output data a determination that the target cancer cells comprise cancer cells using the one or more images obtained using the one or more primary and / or secondary labeling agents.

Images