Patent Application
20140329300
This disclosure relates generally to a device for analysis of a sample, though more specifically, to a slide for culturing, imaging, and/or processing the sample.
Suspensions often include materials of interest that are difficult to detect, extract and isolate for analysis. For instance, whole blood is a suspension of materials in a fluid. The materials include billions of red and white blood cells and platelets in a proteinaccous fluid called plasma. Whole blood is routinely examined for the presence of abnormal organisms or cells, such as fetal cells, endothelial cells, epithelial cells, parasites, bacteria, and inflammatory cells, and viruses, including HIV, cytomegalovirus, hepatitis C virus, and Epstein-Barr virus and nucleic acids. Currently, practitioners, researchers, and those working with blood samples try to separate, isolate, and extract certain components of a peripheral blood sample for examination. Typical techniques used to analyze a blood sample include the steps of smearing a film of blood on a slide and staining the film in a way that enables certain components to be examined by bright field microscopy.
On the other hand, materials of interest composed of particles that occur in very low numbers are especially difficult if not impossible to detect and analyze using many existing techniques. Consider, for instance, circulating tumor cells (“CTCs”), which are cancer cells that have detached from a tumor, circulate in the bloodstream, and may be regarded as seeds for subsequent growth of additional tumors (i.e., metastasis) in different tissues. The ability to accurately detect and analyze CTCs is of particular interest to oncologists and cancer researchers, but CTCs occur in very low numbers in peripheral whole blood samples. For instance, a 7.5 ml sample of peripheral whole blood that contains as few as 3 CTCs is considered clinically relevant in the diagnosis and treatment of a cancer patient. However, detecting even 1 CTC in a 7.5 ml blood sample may be clinically relevant and is equivalent to detecting 1 CTC in a background of about 50 billion red and white blood cells. Using existing techniques to find, isolate and extract as few as 3 CTCs of a whole blood sample is extremely time consuming, costly and is extremely difficult to accomplish.
As a result, practitioners, researchers, and those working with suspensions continue to seek systems and methods to more efficiently and accurately detect, isolate and extract samples of a suspension.
This disclosure is directed to a device and method of using the device to analyze a sample. The sample may be a suspension, a portion of the suspension, a particular component of the suspension, or the like. For example, the suspension may be blood, the portion may be buffy coat, and the component may be circulating tumor cells. The device, such as a slide, may be used to hold the sample for imaging or further processing. The device comprises a cavity that may be sealed on one side by a porous membrane and sealed on an opposite side by a non-porous cover. The porous membrane allows for reagents to be introduced to the sample without diluting the sample. The non-porous cover allows for imaging of the sample. The device further comprises a nozzle for introducing the sample into a space within the cavity.
As shown in
The shelf 106 may be located at any height within the cavity 104. For example, the shelf 106 may be located at a distance from the second side that is equal to the height of the non-porous cover 112, such that when the non-porous cover is placed on the shelf 106, the non-porous cover 112 is flush with the second side of the main body 102. The location of the shelf 106 within the cavity 104, such as by being closer to the second side of the main body 102 than the first side of the main body 102, may also form a chamber above the porous membrane 110 into which reagents, such as fixatives, permeabilizing agents, and/or labeling agents, may be introduced, as the pores of the porous membrane 110 allow for the molecules of the reagents to enter the sample layer. The reagent may be added to the chamber and then aspirated off (or removed by any appropriate method) after an appropriate amount of time has passed to permit the molecules to cross the porous membrane 110 and interact with the sample. Alternatively, the reagent may be introduced between the porous membrane 110 and the non-porous cover 112 with the sample sitting on top of the porous membrane 110.
The non-porous cover 112 may be placed at the second side of the cavity 104 or on a second side of the shelf 106. The non-porous cover 112 provides a window through which the sample may be imaged. The non-porous cover 112 may allow for the use of various magnification objectives, such as up to 100×, including 10×, 40×, 60×, and 63×. The non-porous cover 112 may be transparent or semi-transparent. The non-porous cover 112 may be secured to the shelf 106 by welding, such as ultrasonic, thermal or laser; an adhesive, such as cyanoacrylate, an epoxy, vacuum grease, such as high vacuum silicone grease clips, detents, press fit, or interference fit; or the like. The non-porous cover 112 may be optically clear to permit imaging of the sample within the cavity 104.
The non-porous cover 112 may be composed of glass, crystal, plastic, or combinations thereof. Alternatively, the non-porous cover 112 may be embedded within the main body 102 and, therefore, not removable.
The diameter of the nozzle 308 may be selected to inhibit backflow, thereby causing directional flow. The diameter of the nozzle 308 may inhibit backflow because of the surface tension of the fluid (i.e. the sample or a solution) introduced through the nozzle 308. Alternatively, the nozzle 308 may include a re-sealable port. Alternatively, the nozzle 308 may include a one-way valve to inhibit backflow.
The device 300 may also include grips 312. The grips 312 allow the device 300 to be easily grabbed, manipulated, and transported. A bottom side of the grips 312 each include a a recess 328 to receive the respective grip 312 of another device. The grip-recess connection permits devices to be stacked on top of one another for ease of storage and transportation.
The device 300 may also include an outlet port 314 to remove reagents added to the cavity 304 on top of the porous membrane 316. The porous membrane 316 permits diffusion of the reagents while preventing the sample from leaking out of the porous membrane 316 or being diluted by the solvent or fluid portion of the reagents. The outlet port 314 may be integrated into the main body 302. Alternatively, the outlet port 314 may be integrated into the rim 320 extending up from the main body 302. Alternatively, the reagents may be flowed through the sample and removed via the outlet port 314. The outlet port 314 may include a filter to retain the target analytes within the cavity 304 while withdrawing the reagents. Alternatively, the reagent may be introduced between the porous membrane 316 and the non-porous cover 318 with the sample sitting on top of the porous membrane 316.
Alternatively, the outlet port 314 may be sized and shaped to accept a processing vessel, such as a PCR tube. The processing vessel holds a target analyte of the sample that has been picked and isolated for further processing. The device 300 may also include an insert (not shown) to fit into the cavity 304 to reduce the volume of the cavity 304. The insert (not shown) may be the same shape as the cavity 304 with a cut-out substantially central to the insert (not shown), such that the cut-out is the area in which the sample may be found.
The porous membrane 316 may be removable. For example, the porous membrane 316 may be peeled or pulled off, or, the porous membrane 316 may be attached to a cap that fits within the cavity 304.
The main body may be composed of glass, crystal, plastic, metal, or combinations thereof. The sample may undergo subsequent processing which includes techniques for sequencing, such as nucleic acid sequencing, extracellular analysis and/or intracellular protein analysis such as intracellular protein staining, in situ hybridization (“ISH”), or branched DNA (“bDNA”) analysis.
Alternatively, the main body may include more than one cavity, such as a microtiter plate, such that one or more porous membranes seal one, some, or all of the cavities. When more than one cavity is included, the main body may also include one or more nozzles to be in fluid communication with one, some or all of the cavities.
For the sake of convenience, the sample discussed herein is a buffy coat, though the suspension may be urine, blood, bone marrow, cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid, nipple aspirate fluid, saliva, amniotic fluid, vaginal secretions, mucus membrane secretions, aqueous humor, vitreous humor, vomit, any other physiological fluid or semi-solid, and portions (i.e. plasma or buffy coat from blood) or components (i.e. analytes) of a suspension. Furthermore, the analyte may be a circulating tumor cell (“CTC”), though the target analyte may be a cell, such as ova, a circulating endothelial cell, a fetal cell, a nucleated red blood cell, a vesicle, a liposome, a protein, a nucleic acid, a biological molecule, a naturally occurring or artificially prepared microscopic unit having an enclosed membrane, parasites, microorganisms, viruses, or inflammatory cells.
Returning to
Reagents may be added to the sample layer via the nozzle 114 or inlet port 144 or flowed on top of the porous membrane via the inlet and outlet ports 144 and 146. The reagents, such as antibodies, chemicals to induce changes, permeabilizing agents, fixatives, and/or labeling agents, may be introduced. The pores of the porous membrane 110 allow for the molecules of the reagents to enter the sample layer and interact with the sample. The reagents may include, but are not limited to, fixing agents (such as formaldehyde, formalin, methanol, acetone, paraformaldehyde, or glutaraldehyde), detergents (such as saponin, polyoxyethylene, digitonin, octyl β-glucoside, octyl β-thioglucoside, 1-S-octyl-β-D-thioglucopyranoside, polysorbate-20, CHAPS, CHAPSO, (1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, polyoxyethylene octyl phenyl ether, or octylphenol ethylene oxide), or labeling agents (such as fluorescently-labeled antibodies, enzyme-conjugated antibodies, Pap stain, Giemsa stain, or hematoxylin and eosin stain). The reagents may be removed, such as by aspirating off (or removed by any appropriate method) after an appropriate amount of time has passed to permit the molecules to cross the porous membrane 110 and interact with the sample. Alternatively, when the main body 102 includes the inlet port (not shown) and the outlet port (not shown), the reagents may be removed via the outlet port (not shown). The reagents may be added and removed at different times or simultaneously.
In block 606, the sample may be imaged through the non-porous cover 112 or processed through the porous membrane 110.
The device 100 may be placed on a stage 718 with an aperture 720. The excitation light may be reflected by a dichroic mirror 716 and focused by an objective 704 through the aperture 720 and onto the sample layer, which is the space between the porous membrane and the non-porous cover. The different wavelengths excite different fluorescent markers, causing the fluorescent markers to emit light at lower energy wavelengths. A portion of the light emitted by the fluorescent markers is captured by the objective 704, passed through the dichroic mirror 716, and transmitted to a detector 706 that generates images that are processed and analyzed by a computer or associated software or programs. The images formed from each of the markers may be overlaid when a plurality of fluorescent markers, having bound themselves to the target analyte, are excited and emit light. The sample 710 may then be characterized based on the light emission(s) from the fluorescent marker(s) attached to the sample 710. Alternatively, or in addition to, the sample may be imaged using transmitted light, such as by bright field, dark field, phase contrast, differential interference contrast, or the like.
The sample or a sample portion may be subsequently processed by removing the porous membrane or the non-porous cover to extract the sample or the sample portion; or, the porous membrane may be punctured, such as by a syringe or the like, to remove the sample. These process may include, but are not limited to, extracellular and intracellular analysis including intracellular protein labeling; nucleic acid analysis, including, but not limited to, whole genome amplification followed by next generation sequencing, expression arrays, protein arrays, and DNA hybridization arrays, including genomic hybridization arrays; in situ hybridization (“ISH”—a tool for analyzing DNA and/or RNA, such as gene copy number changes); polymerase chain reaction (“PCR”); reverse transcription PCR; or branched DNA (“bDNA”—a tool for analyzing DNA and/or RNA, such as mRNA expression levels) analysis. These techniques may require fixation, permeabilization, and isolation of the sample prior to analysis. Some of the intracellular proteins which may be labeled include, but are not limited to, cytokeratin (“CK”), actin, Arp2/3, coronin, dystrophin, FtsZ, myosin, spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Akt1, Akt2, c-myc, caspases, survivin, p27kip, FOXC2, BRAF, Phospho-Akt1 and 2, Phospho-Erk1/2, Erk1/2, P38 MAPK, Vimentin, ER, PgR, PI3K, pFAK, KRAS, ALKH1, Twistl, Snaill, ZEB1, Fibronectin, Slug, Ki-67, M30, MAGEA3, phosphorylated receptor kinases, modified histones, chromatin-associated proteins, and MAGE.
A device may also be used to culture, for example, a cell line. The cells may be introduced through the nozzle and nourished with media through the porous membrane. The culture may be kept sterile because of the porous membrane, such as sterility filter.
The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the disclosure. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the systems and methods described herein. The foregoing descriptions of specific embodiments are presented by way of examples for purposes of illustration and description. They are not intended to be exhaustive of or to limit this disclosure to the precise forms described. Many modifications and variations are possible in view of the above teachings. The embodiments are shown and described in order to best explain the principles of this disclosure and practical applications, to thereby enable others skilled in the an to best utilize this disclosure and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of this disclosure be defined by the following claims and their equivalents:
This application claims the benefit of Provisional Application No. 61/818,256, filed May 1, 2013, and Provisional Application No. 61/883,753, filed Sep. 27, 2013.
| Number | Date | Country | |
|---|---|---|---|
| 61818256 | May 2013 | US | |
| 61883753 | Sep 2013 | US |
