The field of the invention relates to processing biological specimens, and more particularly, to isolating and imaging cells of a biological specimen using microfluidics devices.
Medical professionals and technicians often prepare a biological specimen on a specimen carrier, such as a glass specimen slide, and review the specimen to analyze whether a patient has or may have a particular medical condition or disease. For example, a specimen is examined to detect malignant or pre-malignant cells as part of a Papanicolaou (Pap) smear test and other cancer detection tests. After a specimen slide has been prepared, automated systems can analyze the specimen and be used to focus the technician's attention on the most pertinent cells or groups of cells, while discarding less relevant cells from further review.
While Pap smears are well known, they can be difficult to image due to variable sample thickness, among other reasons. To address these issues, cell transfer engines based on “track etched” filter membrane technology have been used to prepare a more consistent single layer of cells that can be applied to a slide, analyzed and imaged. One known automated slide preparation system that has successfully utilized track etched membrane filters is the ThinPrep® 3000 system available from Cytyc Corporation 250 Campus Drive, Marlborough, Mass. 01752. The test using this system is generally referred to as a ThinPrep (TP) Papanicolaou (Pap) test, or more generally, a ThinPrep or TP test.
Referring to
During use, one end of the filter 20 is inserted into the solution 18, and the other end of the filter 20 is coupled through the valve 30 to the vacuum source 40. When the valve 20 is opened, negative pressure from the vacuum source 40 is applied to the filter 20 which, in turn, draws solution 18 up into the filter 20. Cells 16 in the drawn liquid 18 are collected on the face of the filter 20. Referring to
While cell transfer engines based on “track etched” filter membrane technology have been used with effectiveness and provide significant improvements over other known methods, such devices and methods can be improved. In particular, it can be difficult to control the placement and presentation of individual cells 16 on the face of the filter 20. This may present difficulties in controlling the placement and presentation of cells 16 as they are transferred onto the slide 50, thereby making imaging and analysis or testing of the cells more complicated and time consuming.
The ability to determine cell 16 boundaries is important since it allows full cell 16 border definition and the ability to obtain related cellular measurements and data such as cytoplasm area. These capabilities, in turn, allow accurate measurements of an important manual classification metric, namely, the nucleus/cytoplasm ratio which is an important cytological analysis parameter, and which has not been automatically measured in the past.
Further, membrane-based filters 20 do not allow for effective sorting of cells 16 or clusters 17 of cells by size. Additionally, while known preparation systems can be used to prepare specimens that can be stained, such systems may require relatively large volumes of stain and associated cumbersome staining equipment.
Microfluidics has been used recently to manipulate cells. Known microfluidic cell trapping techniques are described in “Cell trapping in Microfluidic chips,” by Robert M. Johann and “Single-Cell Enzyme Concentrations, Kinetics, and Inhibition Analysis Using High-Density Hydrodynamic Cell Isolation Arrays,” by Dino Di Carlo et al. and “Dynamic Single Culture Array” by Dino Di Carlo et al., the contents of all of which are incorporated herein by reference. Johannn describes various immobilization methods including contactless cell trapping and contact-based cell trapping. Di Carlo et al. describe a specific physical barrier that is designed to catch cells based on fluid flowing over an array of cell traps. Other microfluidics systems relate to detecting the presence of certain molecules, e.g., DNA.
While certain microfluidic devices and associated cell manipulation have been proposed, known microfluidic devices and techniques do not provide for effective separation, placement and transfer of cells from a heterogeneous sample of cells that includes other constituents such as lubricants and bodily fluids including blood and mucus. Further, known microfluidic devices do not provide these capabilities on a large scale to provide efficient specimen processing, including preparation and imaging of non-living, preserved specimen samples that are fixed to a substrate for purposes of examination and analysis. Therefore, known microfluidic devices and research are not suitable for cervical cytology and related preparation and analysis of such specimens.
According to one embodiment, a microfluidic apparatus for isolating cells of a cytological specimen includes a substrate and a microfluidic cellular isolation element associated with the substrate. The isolation element includes an outer wall, a channel, a partition member and a receptacle. The outer wall defines an inlet, an outlet, and an isolation element interior, and the channel is defined within the isolation element interior and in fluid communication with the outer wall inlet. The partition member is positioned within the isolation element interior and includes an inner wall that defines an inlet aperture, an outlet aperture, and a partition member interior. The receptacle is positioned within the partition member interior. The isolation element is configured such that fluid introduced through the outer wall inlet flows through the channel in a first direction, the partition member situated such that fluid flows from the channel into the partition member interior through the partition member inlet aperture in a second direction different than the first direction, the receptacle positioned relative to the partition member inlet to catch and retain a cell carried by the fluid.
According to another embodiment, a microfluidic apparatus for isolating cells of a cytological specimen includes a substrate and a microfluidic cellular isolation element associated with the substrate. The isolation element and the substrate are removably attached to each other. The isolation element includes an outer wall, a channel, a partition member and a receptacle. The outer wall defines an inlet, an outlet, and an isolation element interior, and the channel is defined within the isolation element interior and in fluid communication with the outer wall inlet. The partition member is positioned within the isolation element interior and includes an inner wall that defines an inlet aperture, an outlet aperture, and a partition member interior. The receptacle is positioned within the partition member interior. The isolation element configured such that fluid introduced through the outer wall inlet flows through the channel in a first direction, and the partition member situated such that fluid flows from the channel into the partition member interior through the partition member inlet aperture in a second direction different than the first direction. The receptacle is positioned relative to the partition member inlet to catch and retain a cell carried by the fluid. The isolation element and the substrate are removably attached to each other, and a cell caught and retained by the receptacle is located between the substrate and the isolation element.
A further embodiment is directed to a microfluidic apparatus for isolating cells of a cytological specimen that includes a substrate and a microfluidic cellular isolation element associated with the substrate. The isolation element includes an outer wall, a channel, a partition member and a plurality of receptacles. The outer wall defines an inlet, an outlet, and an isolation element interior, and the channel is defined within the isolation element interior and is in fluid communication with the outer wall inlet. The partition member is positioned within the isolation element interior and includes an inner wall that defines a plurality of inlet apertures, an outlet aperture, and a partition member interior, and the plurality of receptacles are situated within the partition member interior. Each receptacle includes a plurality of receptacle components that are separated from each other and arranged to catch a single cell or a cell cluster. The isolation element is configured such that fluid introduced through the outer wall inlet flows through the channel in a first direction, and the partition member situated such that fluid flows from the channel into the partition member interior through the respective partition member inlet apertures in a second direction different than the first direction. The receptacles are positioned relative to the partition member inlet apertures to catch and retain cells carried by the fluid.
A further alternative embodiment is directed to a method of isolating cells of a cytological specimen utilizing a microfluidic cellular isolation element associated with a substrate. The method includes introducing a fluid or solution through an inlet of an outer wall of the isolation element. The introduced fluid flows in a first direction through a channel defined within an interior or inner space of the isolation element. Fluid flows from the channel and through an inlet aperture of a partition member positioned within the isolation element interior in a second direction different than the first direction. The method also includes catching and retaining a cell carried by fluid flowing in the second direction in a receptacle positioned within the partition member. [0017] Another alternative embodiment is directed to a method of isolating and analyzing a cell of a cytological specimen utilizing a microfluidic cellular isolation element associated a substrate. The method includes introducing a fluid or solution through an inlet of an outer wall of the isolation element. The introduced fluid flows in a first direction through a channel defined within the interior or inner space of the isolation element. Fluid flows from the channel through an inlet aperture of a partition member positioned within the isolation element interior in a second direction different than the first direction. The method further includes catching and retaining a first cell in fluid flowing in the second direction in a receptacle positioned within the partition member. The method further includes releasing the first cell (e.g., after analyzing the first cell), and then catching and retaining a second cell in fluid flowing in the second direction to replace the released first cell. The second cell may then be analyzed.
Another alternative embodiment is directed to a method of isolating and imaging a cell of a cytological specimen utilizing a microfluidic cellular isolation element associated with a substrate. The method includes introducing a fluid or solution through an inlet of an outer wall of the isolation element. The introduced fluid flows in a first direction through a channel defined within the interior or inner space of the isolation element. Fluid flows from the channel through an inlet aperture of a partition member positioned within the isolation element interior in a second direction different than the first direction. The method further includes catching and retaining a first cell in fluid flowing in the second direction in a receptacle positioned within the partition member. The method further includes releasing the first cell from the receptacle (e.g., after processing or imaging the first cell), and then catching and retaining a second cell in fluid flowing in the second direction to replace the released first cell. The second cell may then be imaged.
In one or more embodiments, the second direction is substantially transverse to the first direction. Further, in one or more embodiments, a partition member includes multiple receptacles, and the receptacles may be different sizes. A smaller receptacle may be configured to catch and retain a single cell, and a larger receptacle may be configured to catch and retain a cluster of cells. In one embodiment, the smaller receptacle is positioned closer to the outer wall inlet than the larger receptacle. The inlet apertures of the partition member may also be different sizes. A smaller aperture may be configured to allow passage of a single cell, and a larger receptacle sized to allow passage of a cluster of cells. In one embodiment, the smaller inlet aperture is positioned closer to the outer wall inlet than the larger inlet aperture.
In one or more embodiments, an isolation element may include multiple partition members, thereby defining multiple channels, each of which is in fluid communication with the outer wall inlet, and at least one channel being defined between walls of adjacent partition members.
In one or more embodiments, a preconditioning element, e.g., located outside of the isolation element, configured to break apart cell clusters carried in the fluid. The cells and/or remaining clusters may then be caught by one or more receptacles within the isolation element.
Other and further aspects and embodiments are described herein and will become apparent upon review of the following detailed description and drawings.
Referring now to the drawings in which like reference numbers represent corresponding parts throughout and in which:
In the following description, reference is made to the accompanying drawings which form a part hereof, and which show by way of illustration specific embodiments and how they may be practiced. It is to be understood that changes may be made without departing from the scope of embodiments.
Referring to
Embodiments may be used to isolate cells 16 and/or clusters 17 of cells, and reference is made generally to cells 16 unless certain configurations specifically involve isolation of clusters 17 of cells. Further, a solution or fluid as used in this specification is defined as a solution, fluid, material or substance that includes cells 16 or clusters 17 of cells and can flow through the microfluidic cellular isolation element 820. Examples of solutions or fluids 18 that may include cells 16 or clusters 17 of cells include a liquid-based solution, such as PreservCyt available from Cytyc, a gel-based solution, and a bodily fluid. Thus, cells 16 and clusters 17 of cells may be diluted in another substance or fluid (e.g., as in the case of a liquid-based or gel-based solution), or be part of a bodily fluid (e.g., cells of an undiluted specimen sample obtained directly from a cervix). For ease of explanation, reference is made to a solution 18 in the form of a liquid-based solution that flows through a microfluidic apparatus 800, but it should be understood that embodiments can be used to isolate cells in various solutions 18. Further, apparatus, system and method embodiments may be implemented to analyze cytological specimens including cervical specimens and other types of specimens. For ease of explanation, reference is made to cervical specimens in a solution 18.
In one embodiment, the substrate 810 is a glass substrate, such as a glass specimen slide. For example, the substrate 810 may be a known glass slide having a thickness of about 0.05 inch, a width of about 1.0 inch, and a length of about 3.0 inches. The substrate 810 may have a shape and size that is similar to or the same as a known specimen slide so that the substrate 810 can be manipulated by known slide processing systems and stored in known slide receptacles. In another embodiment, the apparatus 800 may be significantly smaller since the isolation element 820 has dimensions on the order of microns. Indeed other substrate dimensions and shapes can be utilized, and
As shown in
The isolation element 820 includes one or more cellular barriers, traps or partition members 822 (generally referred to as partition members 822) that are formed on or in PDMS material 824 using known micro-fabrication/micro-molding methods. The partition members 822 may include micro-fabricated channels, gates and cell receptacles or traps that are defined by the isolation element 820 and formed between the isolation element 820 and the surface 812 of the substrate 810 for catching and holding cells 16 from solution 18 that flows in different directions within the isolation element 820.
In the embodiment shown in
The fluid inlet 831 and the fluid outlet 832 may also be utilized to introduce and carry away other fluids and solutions used for preparing or analyzing a cytological specimen 14. For example, the fluid inlet 831 and the fluid outlet 832 may serve as fluid path for cytological dyes or stains. Alternatively, separate inlet and outlet ports (not shown in
Referring to
Referring to
In the illustrated embodiment, the partition member 822 may be formed to have a rectangular shape, but the partition member 822 may have other shapes and configuration, e.g., square, triangular, diamond, and other shapes depending on the micro-fabrication technique and equipment that is utilized.
In the illustrated embodiment, the partition member 822 includes four sides 920a-d that define an inner space or interior 823. A first side 920a defines one or more inlet apertures or gates 922 (generally referred to inlet aperture 922). One inlet aperture 922 is shown for purposes of explanation, but it will be evident that the side 920a may define other numbers of inlet apertures 922. A top or downstream side 920b may define an outlet aperture 924. In the illustrated embodiment, the sides 920c and 920d are solid and do not define inlet or outlet apertures. Thus, in the illustrated embodiment, the partition member 822 may have a certain side 920a that defines only inlet apertures 922, a certain side 920b that defines only an outlet aperture 924, and certain sides 920c,d that are solid and define no apertures.
A micro-fabricated fluid channel 930 in fluid communication with the inlet 914, which is in fluid communication with the fluid inlet 831, is defined between the first side 920a of the partition member 822 and the outer wall 910. The base member 810 may have a thickness of about 6 mm, the isolation elements in 820 are molded within the base member 810, and the fluid channel 930 may be about 70 to about 100 microns wide, and about 40 microns in depth. In the illustrated example, the channel 930 extends around the corner of the partition member 822 defined by sides 920a,b. Solution 18 having cells 16 of the specimen 14 is introduced from the fluid inlet 831 and through the inlet 914. From the inlet 914, solution 18 flows downstream through the channel 930 along side 920a of the partition member 822.
The solution 18 initially flows through the channel in a first direction 941 (generally represented by an arrow parallel to the channel 930), otherwise referred to as laminar flow, or flow of solution 18 without turbulence. Laminar flow 941 within the channel 930 provides for the flow of solution 18 in a relatively predictable manner. A portion of the solution 18 flowing in the first direction 941 and through the channel 930 changes direction and flows through an inlet aperture 922 defined by the first side 920a of the partition member 822 (e.g., due to a pressure differential and/or surface adhesion). This is otherwise referred to as lateral flow, or flow in a second direction that is different than the first direction (generally represented by arrows that are not parallel to the arrow 941 or the channel 930). According to one embodiment, the isolation element 820 is fabricated so that the flow of solution 18 in the second direction 942 is substantially transverse or perpendicular to the laminar flow through the channel 930 in the first direction 941. According to one embodiment, the second direction 942 is at an angle of about 45 to 90 degrees relative to the first direction 941.
An individual cell 16 or a cluster 17 of cells carried by the solution 18 may be captured by a cell receptacle 950 positioned within the partition member 822 after the solution 18 flows through the inlet aperture 922 and towards the receptacle 950. For this purpose, the receptacle 950 may be arranged at a corresponding angle so that the open or receiving end of the receptacle 950 faces the inlet aperture 922 and is in the path of solution 18 flowing through the inlet aperture 922 in the second direction 942.
Solution 18 that enters the partition member 822 and flows past the receptacle 950 may continue to flow downstream through the interior 823 of the partition member 822, and exit the partition member 822 through the outlet aperture 924, where it may be re-combined with solution 18 that did not enter the partition member and flowed through the channel 930 around sides 920a,b. The solution 18 may then continue to flow downstream towards the outlet 916 and through the fluid outlet 832.
This micro-fabrication structure and the flow of solution 18 in different directions is beneficial since the lateral flow of solution 18 in the second direction 942 through the inlet aperture 922 minimizes clogging of the inlet apertures 922 by constituents such as lubricants and bodily fluids including blood and mucus. Further, the flow of solution 18 in the first direction 941 inhibits clogging of the inlet apertures 922 by flushing away particles that are too large to pass through the inlet apertures 922, while allowing particles (such as individual cells 16 or cell clusters 17) that are sufficiently small to pass through and traverse the inlet apertures 922 and lodge into a receptacle 950.
More particularly, as shown in
During use, solution 18 including cells 16 of a specimen 14 flows transversely in the second direction 942 through the inlet aperture 922 and towards the receptacle 950, which captures the cell 16 as shown in
For example, a cell receptacle 950 as shown in
Referring to
One example of a partition member 822 constructed in accordance with one embodiment for isolating individual cells 16 includes about 100 inlet apertures 922 and about 500 receptacles 950 within the partition member 822. The partition member 822 may have a width of about 500 microns and a height of several millimeters. Each inlet aperture 922 may have a width of about 75-200 microns, and the spacing between inlet apertures 922 may be about 100 microns. It should be understood that various other numbers and configurations of inlet apertures 922, receptacles 950 and partition members 822 may be utilized and may vary as necessary to capture a desired number of individual cells 16.
Referring to
One example of a partition member 822 constructed in accordance with an embodiment for isolating clusters 17 of cells includes about 25 inlet apertures 922 and about 125 receptacles 950 within the partition member 822. The partition member 822 may have a width of about 500 microns, a height of several millimeters, each inlet aperture 922 may have width of about 200 microns, and the spacing between inlet apertures 922 may be about 100 microns. It should be understood that various other numbers and configurations of inlet apertures 922, receptacles 950 and partition members 822 may be utilized and may vary as necessary to capture a desired number of clusters 17.
In other embodiments, an isolation element 820 may include multiple partition members, e.g., multiple partition members arranged side-by-side in an array with corresponding sides 920a-d. With this configuration, a corresponding array of cells 16 and/or clusters 17 of cells may be captured and processed. For example, referring to
In the illustrated configuration, inlet apertures 922 are formed on the same side 910a of each partition member 822. A first channel 930a is defined between the side 920a of the partition member 822a and the outer wall 910 (as shown in
Referring to
Referring to
According to one embodiment, referring to
In a further embodiment, a cell 16 that is captured within a cell receptacle 950 may be released, and another cell 16 can be captured to replace the released cell. For example, cells 16 that were initially captured by receptacles 950 may be imaged and examined. A cytotechnologist may then determine that certain cells 16 may be abnormal or suspicious, in which case these cells 16 may be retained, whereas cells 16 that are determined to be normal can be released and replaced with other captured cells 16 for examination. In this manner, a larger number of cells 16 are reviewed to provide a more thorough and accurate analysis by releasing and replacing normal cells 16 with other cells 16 that may be abnormal or suspicious. Release of captured cells 16 can be carried out using known mechanical, optical or electronic techniques, e.g., as described in “Cell trapping in Microfluidic chips,” by Robert M. Johann, the contents of which were previously incorporated herein by reference.
Cells 16 and cell clusters 17 that are isolated using the isolation element 820 mated or attached to the substrate 810 may be directly imaged as is, i.e., without having to transfer collected cells 16 and clusters 17 to another substrate or specimen slide since the captured cells 16 and clusters 17 may be visible through the substrate 810 and/or the isolation element 820. This capability provides enhanced automated reviewing of specimen samples having full cell border definitions so that measurements (such as cytoplasm area) can be completed and allow key manual classification metrics (such as the nucleus/cytoplasm ratio) to be automatically measured. Alternatively, the collected cells 16 and clusters 17 may be transferred from the isolation element 820 to another substrate, such as a glass specimen slide, and the transferred cells 16 and clusters 17 may then be imaged using various known cytological imaging systems.
Images of the isolated cells 16 and cell clusters 17 generated by the optical stack are provided to the computer 2102 for analysis. After images of the isolated specimen cells 16 and cell clusters 17 are acquired, the images are processed to identify or rank cells and cell clusters that are of diagnostic interest. In some systems, this includes identifying those cells that most likely have attributes consistent with malignant or pre-malignant cells and their locations (x-y coordinates) on the slide. For example, the processor 2102 may select about 20 fields of view, e.g., 22 fields of view, which include x-y coordinates identifying the locations of cells 16 and cell clusters 17 that were selected by the processor 2101. This field of view or coordinate information is provided to a microscope, which steps through the identified x-y coordinates, placing the cells or clusters of cells within the field of view of the technician.
Although particular embodiments have been shown and described, it should be understood that the above discussion is not intended to limit the scope of these embodiments. Various changes and modifications may be made without departing from the spirit and scope of embodiments. For example, dimensions of various components are provided for purposes of explanation, and the sizes of components of embodiments may vary as necessary. Additionally, embodiments of microfluidic cell isolation devices may include various numbers of isolation elements, and each isolation element may include different numbers and configurations of partition members, inlet apertures, outlet apertures and cell receptacles. Further, embodiments may be implemented to capture only individual cells, only cell clusters, or a combination of individual cells and cell clusters as needed using cell receptacles of various shapes and sizes and having various numbers of receptacle elements. Embodiments may also be adapted or applied to isolating and analyzing cells of other types of specimens besides cervical specimens, and specimens may be in various solutions. Further, although embodiments are described with reference to micro-fabrication and hydrodynamics, embodiments may also be implemented using other microfluidic techniques including optical or dielectrophoretic techniques. Thus, embodiments are intended to cover alternatives, modifications, and equivalents that fall within the scope of the claims.
The present application claims the benefit under 35 USC §119 of provisional application Ser. No. 60/975,070, filed Sep. 25, 2007. The aforementioned application is hereby incorporated by reference herein in its entirety.
Number | Date | Country | |
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60975070 | Sep 2007 | US |