Position-assisted negative particle pass / fail (PANR) for sorting and enrichment of targeted cells

Abstract

A novel method and apparatus for sorting and enriching target cells of interest from a mixture.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62,837,769, filed April 24, 2019, which is incorporated herein by reference. [Background technology]

[0002] Sorting and enrichment of cells and particles of interest from mixtures is an interesting technique in many fields (Robinson and Roederer 2015; Shields IV, Reyes et al. 2015; Gabriel, Calleja et al. 2016). Multiparameter single-cell sorting is important in many fields, but is too slow for many important applications [1-4]. Thus, techniques that rapidly enrich for subsets of cell types based on their physical properties (e.g., size, density, compressibility) are often used as the first step of multiparameter sorting to speed up the entire cell separation process [2, 4-6]. However, adding an enrichment step not only increases the chance of losing rare cells, but also increases the complexity and cost of the sorting process [5]. What is needed is an approach that allows for fast multiparameter cell and particle sorting, regardless of cell and particle size.

[0003] For the purposes of the present invention, the term "sorting" refers to the highly specific separation of cell or particle types, one by one, based on cellular or particle characteristics whose identification usually requires some biological or physical marker. Also, by definition, "enrichment" refers to the relative enrichment of a given cell or particle type with respect to a starting mixture of cells or particles. Finally, in brief, the present invention is equally applicable to other particles in aqueous fluids, such as microspheres, multicellular aggregates, small organisms, nanoparticles (such as viruses or prions), biological nanoparticles, etc.

[0004] Because most cells display similar physical properties, the enrichment process is a fast process but does not provide high purity [2]. Enrichment processes such as RBC lysis for WBC enrichment [7] and micro-post arrays for WBC separation [8] have been successful because of the significant physical differences between RBCs and WBCs. Because of the efficiency of such approaches, the development of similar techniques to isolate disease-indicating target cells has become a highly sought-after goal [9-12]. Although some diseases (e.g., malaria) cause damage that allows for efficient physical separation [6], most do not alter cell properties sufficiently to allow for high-purity enrichment from background cells that display diverse physical properties [6]. Furthermore, for homotypic cells, the physical changes from one individual cell to the next are significant, which means that systems that rely solely on physical properties for cell sorting or cell enrichment are more likely to view cells of different types as more similar than two cells of the same type.

[0005] Nonetheless, many enrichment approaches have been pursued, including deterministic lateral displacement[13-15], inertial focusing[16-20], acoustophoresis[21-23], dielectrophoresis[24-26], and countless others. Although the label-free nature and throughput of these approaches are desirable, no enrichment approach provides purity comparable to that of multiparameter sorting.

[0006] Another approach, magnetically activated cell sorting (MACS), involves the binding of magnetically tagged antibodies followed by magnetic capture and cell sorting [27, 28]. MACS is a fast, quiet and high-throughput operation, but it does not allow the isolation of high purity cells of most rare cell types, which require several cell markers for their identification. The only way to perform multiparameter sorting by MACS requires successive washing / sorting steps, because each sort is a binary operation

[29] .

[0007] In comparison, fluorescence-activated cell sorting (FACS) allows cells to be sorted simultaneously using fewer than 20 cell markers [30, 31]. Using FACS, relatively rare cells (usually identified by fluorescent antibodies) can be isolated with near 100% purity in a matter of hours [30, 31]. These properties have made it the mainstream technique for separating cell populations [30-32]. In FACS, optical analysis is performed as the cells pass through the flow, and cells of interest are labeled to emit light at specific wavelengths after excitation with a powerful light source

[32] . The ability to simultaneously identify multiple colors of fluorescence allows cells to be identified by the type of molecule they express or display, and is often the only way different cell types can be differentiated [30, 31]. Each cell can be identified in real time as it passes through the system, generating a sorting signal and redirecting the cells of interest using the sorting mechanism

[32] . A typical sorting mechanism uses a pressure-driven device to induce droplet formation as the high-velocity fluid exits the nozzle. Only droplets containing the desired cells are electrostatically charged, and this charge is used to redirect the droplets while passing through an oppositely charged plate, allowing successful cell sorting. Due to the small droplet volume and high speed operation of FACS, this approach allows sorting to be performed at a rate of 50,000 cells / s

[32] . In contrast, recent advances in microfluidics have instead allowed the use of valves that open and close at 30 KHz, achieving nearly matching sorting speeds [33, 34]. However, the use of hydrodynamic focusing and / or microfluidics in these commercial systems results in sample delivery flow rates below 100 mL / min, making it impossible to sort out large samples. For this reason, enrichment steps are performed before fluidic sorting of rare cell populations. Unfortunately, such steps not only increase complexity, but also prolong processing times and cause loss of rare cells, all of which make the isolation of rare cells from large samples highly problematic.

[0008] For particles larger than a single cell (i.e., particles larger than 50 mm in diameter), the choice of sorting technique becomes more limited. Techniques exist for sorting large particles that are similar to FACS described above, but use a puff of air to displace a jet in the air stream to sort [35, 36]. Due to limitations in hydrodynamic focusing of large particles and the use of relatively slow sorting mechanisms, this approach can only sort particles up to 200 mm in diameter at about 100 / s, and even slower for larger particles.

[0009] For these reasons, approaches that can accurately separate cells and other particles in a simple, fast, yet gentle manner are a worthy goal. Most of the proposed improvements employ microfluidic solutions, and a number of microfluidic cell sorting devices are commercially available

[37] . Nevertheless, cell sorting volumetric flow rates are limited to about 100 μ / min, and effective sorting speeds are only about 50K cells / s (and even lower for larger particles). This is a significant limiting factor for the sorting of large numbers of rare cells (e.g. stem cells for therapeutic purposes), as these approaches are unable to analyze enough samples to produce large quantities of rare cell populations in a reasonable time.

[0010] Despite this research and development, there are fundamental limitations to fluorescence-activated cell sorting. Most currently employed approaches use microfluidics and extremely fast valves to make each sorting step as fast as possible and in the smallest possible volume. An inherent feature of this approach is that sorting speed is limited to approximately 50,000 events / s due to the stochastic arrival of particles and the exponential increase in system pressure as linear velocity increases.

[0011] Due to these limitations, alternative sorting approaches have been explored. For example, ensemble decision aliquot ranking (EDAR) uses lasers to generate fluorescent signals from many cells in an aliquot of a sample in a microfluidic channel, but this dramatically reduces the ability to resolve true positive signals from signals generated by background binding of many antibody types to cells coexisting in the aliquot [38, 39]. After analysis, the aliquot flows from both sides into a junction that becomes an input buffer, and the incoming cell aliquot is focused into the central outlet of a three-way outlet. A solenoid valve is used to control the flow of the input buffer, allowing the aliquot to be diverted by EDAR into one of the side outlets. In addition to EDAR, a number of other approaches have been proposed, including the use of piezoelectric actuators to switch flows or cells [40-42] and lasers to move cells

[43] , but all of these have limitations in throughput and sorting speed.

[0012] Thus, there remains a great need for systems that can rapidly and accurately select and / or enrich for cells of interest in mixed samples or populations. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Piyasena, ME and SW Graves, The intersection of flow cytometry with microfluidics and microfabrication. Lab on a Chip, 2014. 14(6): p. 1044-1059. [Non-Patent Document 2] Shields IV, C.W., C.D. Reyes, and G.P. Lopez, Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation. Lab on a Chip, 2015. 15(5): p. 1230-1249.

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[0014] The present invention, according to various embodiments, provides novel methods and apparatus that allow for the sorting and enrichment of target cells of interest from a mixture. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram illustrating a PANR system according to an embodiment of the present invention. [Figures 2A-2E] 2A-2E are schematic diagrams illustrating a non-limiting exemplary device in use for detecting, sorting and enriching target cells of interest. [Figure 2A] FIG. 2A shows how a cell is positioned by an acoustic standing wave generated by a PZT driver. [Figure 2B] FIG. 2B shows a target cell appearing in the scrutiny area. [Figure 2C] FIG. 2C illustrates the signals generated by the detection system and the valve opening of the reject flow path. [Figure 2D] FIG. 2D shows target cells entering the valve openings of the recovery and reject channels. [Figure 2E] FIG. 2E shows the system with the valves returned to an open state. [Diagram 3]FIG. 3 is a flow diagram illustrating iterative PANR according to one embodiment of the present invention. [Figure 4] FIG. 4 shows the concentration in the collection channel (CC) as a function of the target cell concentration (C+) and background cell concentration (C-). [Diagram 5] FIG. 5 shows the volume in the collection channel (VC) as a function of target cell concentration (C+) and background cell concentration (C-). [Figure 6] Figure 6 shows the fractional purity in the collection channel as a function of target cell concentration (C+) and background cell concentration (C-). Note that if C+>>C-, there will be more than one target cell at each sort, resulting in a fractional purity of 2 or more. This condition is not shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In one embodiment, the present invention provides a novel method and apparatus for selecting and enriching target particles of interest from a mixture, which, in accordance with various aspects described below, will be referred to as Positionally Assisted Negative particle Rejection (PANR).

[0017] FIG. 1 is a schematic diagram of a PANR system according to one embodiment of the invention. The system 10 generally comprises a flow channel 12 bounded at a distal end to form a collection channel 14, and a rejected cell channel 16. The intended flow direction is indicated by arrow 18. (Note that the terms "proximal" and "distal" are used herein to refer to the intended flow direction in a standard operating system where a fluid sample typically flows from the proximal end of the channel to the distal end.) The proximal end of the channel 12 comprises a particle positioning and focusing device 20. The particle positioning device positions (or focuses) the particles in the channel 14. In PANR, once the particles have been positioned, two tasks can be performed: the first is to position each particle and determine this with a detector 22, and the second is to position each particle and direct it to the rejected cell channel 16 unless otherwise indicated. The detector 22 is in communication with a valve 24.

[0018] In operation, particles within a sample are introduced into the proximal end of the collection channel and positioned (focused) by device 20. For example, device 20 may be any device capable of positioning particles and performing the first and second tasks described above. Devices such as piezoelectric transducer (PZT) actuated devices capable of generating acoustic standing waves may be used. Other devices and positioning methods that may be used include, but are not limited to, micropost arrays for deterministic lateral displacement, inertial focusing, dielectrophoretic focusing. Additionally, positioning methods that require the addition of fluids, such as hydrodynamic focusing, are also valid as long as all of the added fluids are accounted for in the calculations and approach.

[0019] Once focused, the particles are interrogated by a detection device 22. Generally, the detection device 22 can distinguish a single particle or a very small number of particles of interest from background particles (see, e.g., refs. 44-48). In one specific embodiment, the detection device 22 comprises a focused light source, such as a laser and an optical detector. In this embodiment, once the particles are positioned, they are allowed to flow into the interrogation zone 26, where they encounter the focused light and generate an optical signal that is analyzed by the optical detector to determine which particles are particles of interest. Of course, any method of cell interrogation (imaging, ultrasound, etc.) can be used as long as it is capable of identifying cells of interest with the optical signal being detected and generating a signal upon detection of the cells of interest.

[0020] In this case, the detector 22 is in communication with a valve 24. In normal operation, if no particles of interest are detected, the valve 24 opens and the sample fluid flows out of the detector channel and into the rejected particle channel 16 and the return channel 14. It is important to note that since the positioning device 20 specifically positions all particles in the channel 12, if any particles are present in the detector channel, they will naturally flow into the rejected particle channel 16 (which may be referred to as the exhaust or drain channel). This is also true if a smaller volume of fluid (not containing any particles) flows into the return channel 14.

[0021] In contrast, if the detection device 22 identifies a particle of interest, it sends a signal to the valve 24 to close the valve, which causes the particles in the scrutiny zone to flow outwardly towards the collection channel 14. If desired, the collected particles of interest (along with any stray background particles) may be recirculated back to the proximal end of the device, as indicated by dashed arrow 28, or may be subjected to another or further selection / enrichment cycle.

[0022] The channels and valves can be constructed from standard materials commonly used in the fluidics and microfluidics industries and can be manufactured using standard techniques, including, but not limited to, micromachining, microlithography, etching, and 3D printing.

[50] Suitable solenoid valves can be manufactured or commercially available; for example, commercially available solenoid valves with a response time of 200 μs are available from TLX Technologies (Pewaukee, WI).

[0023] As will be explained in more detail below, while FIG. 1 shows a single flow path that may or may not automatically recirculate collected particles of interest, the present invention includes configurations in which multiple flow paths may be used in parallel and / or sequential order to rapidly process large volumes of sample. Such multiple flow path configurations may also allow for the detection and collection of one or more particles of interest. In addition, more than one particle of interest may be identified as a particle of interest by the detection device 22. Thus, the system according to various embodiments may be designed to identify and collect two or more specific particle types simultaneously (i.e., in the same flow path), in parallel (i.e., in multiple flow paths operating simultaneously), and sequentially (i.e., detecting one type of particle in a first flow path and another type of particle in a second flow path).

[0024] 2A-2E are schematic diagrams illustrating non-limiting exemplary layers that, in use, detect, sort, and enrich target cells of interest. A fluid sample containing both background cells (black circles) and cells of interest (light circles) enters the left (proximal end) of the channel and flows to the right as indicated by the arrows. In FIG. 2A, cells are shown being positioned by an acoustic standing wave generated by a PZT actuator. Once positioned, the cell flows through the laser beam and an optical signal is emitted that is analyzed by a detector. Whether a particular cell is a cell of interest is determined by analysis of the emitted optical signal. As shown, while only background cells are being detected, the rejected cell channel valve is open and all cells flowing through the device are directed out of the rejected cell channel. Meanwhile, a small amount of fluid (containing no cells) flows into the recovery channel. In this "valve open" state, the amount of fluid directed and flowing into a given channel is determined by the relative cross-sectional area of ​​the channel opening to the total area of ​​the channel, as will be described in more detail below.

[0025] Figures 2A-2E show only one example of several possible design configurations, including, for example, a single valve design or a multiple valve design in which only fluid flows through the collection channel after the target cells are identified.

[0026] Returning to the configuration shown in Figures 2A-2E, when a cell of interest reaches the interrogation area (Figure 2B), the detection system issues a signal and the valve in the rejected cell channel closes (Figure 2C). In this "valve closed" state, flow stops in the rejected cell channel and all flow is forced into the open recovery channel. Forcing all flow into the recovery channel ensures that all cells and all fluids are directed to the recovery channel. When it appears that a cell of interest has entered the recovery channel, the valve begins to open (Figure 2D) and the system returns to the open valve state (Figure 2E).

[0027] In this manner, all of the target cells are collected in the collection channel. Of course, if the valve remains closed long enough, background cells will also be collected. However, if the target cells are relatively few in number compared to the background cells (as described in more detail below), most of the background cells will be diverted to the rejected cell channel, and all of the target cells will be retained in the collection channel. This effectively increases the number of target cells relative to background cells.

[0028] Intuitively, this process results in an increase in the number of cells of interest, and if this approach is used repeatedly (i.e., the harvested volume is returned to the system), eventually only the target cells will remain in the harvest stream. In other words, the first time the system is run, the number of target cells will increase, and if it is repeated, the sorting efficiency will increase and only the target cells of interest will remain in the harvested volume. Of course, at the end of the iterative process, the target cells will be less concentrated due to the increased volume, at which point cells can be harvested at any desired concentration using very simple cell enrichment techniques such as centrifugation or acoustophoresis.

[0029] Therefore, repeated use of PANR (hereafter referred to as rPANR) can effectively select target cells. Figure 3 shows a flow diagram of this approach.

[0030] Application of the systems and methods of the present invention allows for multiple rapid fractionation cycles of target cells, emphasizing total sample recovery while maximizing the fluidic throughput of the system. Additional sorting steps can be rapidly performed to recover target cells in their natural sample context. As described below, this paradigm shift occurs, allowing rPANR to sort cells from as little as a liter of sample, with 10 cells sorted in an automated manner in a few hours. 7As few as one target cell per liter can be recovered in a background of negative cells. Furthermore, the process is not significantly affected by particle size. This sorting speed is not easily achieved with other approaches and has significant implications for rare cell sorting, high-throughput cell sorting and large particle sorting. rPANR itself is of great value across all aspects of biomedical and clinical research, as well as for therapeutic-scale cell separation.

[0031] The PANR process can be described by a set of equations to mathematically model the rPANR process. Equation 1 describes the delay volume (V D ) is calculated, which is the amount of sample that flows into the collection channel due to valve closure, F is the volumetric flow rate (F), and f1t is the time required to open and close the valve. [Formula 1] TIFF0007656332000001.tif8126

[0032] The faster the valve operation speed, the smaller this flow rate becomes. D The less nonspecific background cells present in the channel, the greater the enrichment of the individual PANR pathways. The fraction (U) of the total channel cross-sectional area that is comprised by the collection channel is calculated according to Equation 2, where xArea collection is the cross-sectional area of ​​the collection channel and xArea rejection is the cross-sectional area of ​​the channel for rejected cells. [Formula 2] TIFF0007656332000002.tif15126

[0033] The delay volume (Y) at which the valve closes is calculated using Equation 3. + is the concentration of target cells, and C - is the background cell concentration, and V T is the total sample volume. [Formula 3] TIFF0007656332000003.tif15170

[0034] The delay volume (N) when the valve is opened is calculated using Equation 4. N is the total number of times the valve is opened and closed V T / V D It is calculated by simply subtracting Y from [Formula 4] TIFF0007656332000004.tif37170

[0035] These constants are used to calculate the system variables for the specific model conditions. For example, Equation 5 is used to calculate the volume of the PANR that flows into the recovery channel during one cycle (V C That is, the number of times the valve opens (Y) is counted, and this is multiplied by the delay volume (VD). [Formula 5] TIFF0007656332000005.tif8170

[0036] Add the amount of fluid that is transferred to the recovery channel while the valve is open. This is the amount of fluid that is transferred to the recovery channel when the valve is open (V D ), which is the number of times the valve opens (N) corrected for the fraction of the volume that flows into the collection channel when the valve opens (U). Substituting equations 3 and 4 into equation 5, we obtain equation 6, which gives the total volume of the sample (V T ) the amount that is expected to flow into the recovery channel can be calculated. [Formula 6] TIFF0007656332000006.tif21170

[0037] In addition, as described by Equation 7, the recovery flow path (P C ) to calculate the number of cells that will be finally collected. This corresponds to a low target cell concentration, and the background cell count is calculated as V D The number of valve closures (Y) is calculated as the number of valve closures added to the average number of background cells in the [Formula 7] TIFF0007656332000007.tif14170

[0038] As the target cell concentration increases, two or more cells are bound to the V DThe model itself is likely to be present at low target cell concentrations (e.g., C + :50.2 cells / V D ), which is less than 1 target cell / V D (This means that there is a 98.2% chance that P c can be calculated. [Formula 8] TIFF0007656332000008.tif30170

[0039] Equation 9 combines Equation 6 and Equation 8 to give the concentration of cells in the collection channel (C c ) can be calculated. [Formula 9] TIFF0007656332000009.tif33170

[0040] Finally, the model determines the recovery flow path (P c The total number of target cells of interest (P + ) can be calculated as a function of the total sample volume at the start of the process (V T ) by the known target cell concentration to obtain P + can be calculated.

[0041] Using Equation 8 and Equation 10, the fractional cell purity (Purity) can be calculated (Equation 11). [Formula 11] TIFF0007656332000010.tif30170

[0042] This model allows the V of a configurable system D Useful predictions can be made using real parameters such as the effect of valve response time on V. Using a commercially available miniature solenoid valve (TLX Technologies, Pewaukee, WI) with a response time of 200 μs, one can assume an f1t of 1 ms, which includes the time to send the sort signal to the valve. Using a flow rate (F) of 1 mL / min or 167 μL / s, VD would be 17 nL. <1.2 × 10 4 The target cell number / ml is 1 cell / 5 x V D (85nL) C + This ensures that the low cell concentration required for the model of the present invention can be obtained. + Above this, V D In the case of conventional sorting devices, this is problematic, but the PANR operation allows for more efficient enrichment, although with some limitations. In other words, a larger proportion of target cells will be present in the collection channel. In the present model, the valve is assumed to be open most of the time. The system is designed to operate in a V-shaped flow path during sorting. D V preceding D The amount of target cells contained in the volume of the V D is occupied by target cells, the valve will remain closed. Similarly, Poisson statistics suggest that 4 17 nLV target cells / mL during sorting D 82% of the time volume of 17 nL prior to the injection is free of target cells. In fact, 6 × 10 4 At high concentrations of 100 target cells / mL, the V D contains no target cells 50% of the time, close to the operational limit of the PANR. In terms of channel size, if we use a 100 μm x 100 μm square channel as the lower dimensional limit, the channel will be 1.7 mm long and contain 17 nL. If the cells are positioned in a linear fashion, and assuming a system with a cell diameter of 10 μm, and the cells are packed as beads on a string, this limits the cell concentration to 170 / 17 nL, resulting in a maximum total cell concentration of 1 × 10 7 At these concentrations, the coincidence of some events during analysis can be expected if acoustic cytometry is in use

[49] , but this should not be considered a limitation inherent to optical analytical approaches.

[0043] In the case of Figure 4, the target cell concentration (C +) and background cell concentration (C - ) as a function of the collection channel (C c As can be seen from this plot, the concentration of cells at high cell concentrations (10 4 of target cells / ml and 10 7 If we start the process with a background of 10 cells / ml, the improvement obtained using this approach is very small. In fact, the expected concentration is 10 4 / 10 7 From 10 4 / 7.6×10 6 The improvement was only ~24% enrichment in concentration (i.e., 24% reduction in background cells). This issue was resolved by lowering the starting target cell concentration. 3 and 10 7 If the starting concentration is 10, the enrichment rate is 83%. 2 and 10 7 As shown in Figure 5, if the starting concentration is 10 4 With 10 target cells, ~7.3 mL goes into the collection channel, and 10 3 Using , ~4.5mL goes to the collection channel, and 10 2 Starting from 10 mL, the volume approaches 4 mL, which is consistent with the fractional volume recovered in the model (40%) and the starting volume (10 mL). In Figure 6, we consider the cell count (positive cells / total cells), and as can be seen, the volume approaches 10 mL. 7 10 in background cells 4 Starting with 10 positive cells, the improvement in cell purity is only 0.13%, and remains relatively constant as the target cell concentration decreases (0.12% for 103 / mL and 102 / mL). However, when the relative starting target cell concentration is increased to 100% higher than the background cell concentration (e.g., 10 4 and 10 6 This situation improves dramatically, by a factor of 10, when the starting background cell concentration is closer to 10 5 ~10 4 cells / ml, the ability to achieve a fractional purity of 1 for the target cells of interest is <10 4 It is revealed for the target cell population at a concentration of cells / ml.

[0044] According to one embodiment, the rPANR process of the present invention is carried out using a commercially available Velocyte RT-PCR kit capable of analyzing cell samples at high concentration, an analysis rate of 2×10 cells / s, and a flow rate of 10 mL / min. TM This can be performed using a parallel acoustic flow cytometry system such as the rPANR system (BennuBio, Inc. Albuquerque, NM)

[48] . With such a system, 10–15 parallel streams of various flow cell configurations can be analyzed, ultimately providing up to 16 optical parameters. By performing the rPANR process on a parallel acoustic flow cytometry system, the detection of 107 background cells / mL to 10 -3 ~10 4 Target cell concentrations of up to 100 target cells / mL can be determined by the method of the present invention. Current sorters require a pre-enrichment step in order to sort such samples. In Figure 4, we consider using the 10-stream Velocyt system as the basis for the rPANR process and estimate how it compares to conventional flow sorting with a pre-enrichment step.

[0045] In another embodiment, the detection system and method of the present invention allows for multi-parameter analysis of each cell, i.e., multi-parameter fluorescence, refractive index, and scattering analysis, and identification of cells by optical signals generated by one or more lasers. In addition, when other parameters such as cell impedance, ultrasound response, etc. are used in combination, a sorting signal can be generated based on the analysis of multiple cell characteristics.

[0046] The specific methods and configurations described above represent preferred embodiments, are illustrative, and do not limit the scope of the present invention. In consideration of this specification, other objects, aspects, and embodiments may be conceived by a person skilled in the art, and all of these are encompassed within the scope of the invention as described in the claims. It should be obvious to a person skilled in the art that various substitutions and modifications can be made without departing from the scope and spirit of the present invention. The invention exemplified in this specification can be practiced without elements or limitations that are not specifically disclosed as essential in this specification. The methods and processes exemplified in this specification can be practiced in different sequences of steps, and are not necessarily limited to the sequences of steps disclosed in this specification or described in the claims.

[0047] The terms and expressions used above are for illustrative purposes and are not intended to be limiting, and when such terms and expressions are used, the equivalents of the illustrated and described features and feature parts are not excluded, but various modifications and the like can be implemented within the scope of the present invention. Thus, the present invention has been specifically described with reference to preferred embodiments and features to be used as appropriate, but the ideas disclosed in this specification can be partially modified or changed by those skilled in the art, and such partial modifications and changes are considered to be within the scope of the present invention described in the claims.

[0048] All patents and publications listed below and / or mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each patent and publication is hereby incorporated by reference to the same extent as if it were individually incorporated in its entirety or in its entirety. Applicant reserves the right to actually utilize any disclosures and information contained in any such cited patents or publications. [Explanation of symbols]

[0049] 10. System 12 Distribution channel, flow channel 14 Recovery channel, channel 16 Rejected cell flow path, rejected particle flow path, non-compliant particle flow path, flow path 20 Focusing device, device, positioning device 22 Detectors, detection devices 24 valves 26 Scrutiny Zone 28 Dotted Arrow

Claims

1. A flow path having an inspection zone and an end branching into a rejected particle path and a recovery path having a valve; a positioning device for positioning particles in the flow path such that, when the valve is open, the particles in the flow path will flow into the rejected particle path but will not flow into the return path; and a detector for detecting particles within the inspection zone and in communication with the valve; upon detection of a target particle in the examination zone by the detection device, the valve closes, forcing the target particle and all fluid in the flow path into the collection path; A system for selecting and / or concentrating target particles of interest in a fluid sample, characterized in that a portion of the fluid sample not containing the particles flows into the collection path when the valve is opened, and an inlet is provided upstream from the examination zone for returning a flow containing the collected particles to the flow path, the inlet being fluidly connected to the collection path.

2. The system of claim 1, wherein the positioning device uses acoustic standing waves to position the particle within the flow path.

3. The system of claim 1, wherein the positioning device uses inertial focusing to position the particle within the flow path.

4. The system of claim 1, wherein the positioning device uses dielectrophoretic focusing to position the particle within the flow path.

5. The system of claim 1, wherein the positioning device is a piezoelectric transducer.

6. The system of claim 1, wherein the detection device is an optical detection device.

7. The system of claim 1, wherein the detection device comprises a laser beam through which the particle passes, and the detection device detects an optical signal generated by the particle.

8. The system of claim 1, wherein the detection device uses imaging technology to detect the target particle of interest.

9. The system of claim 1, wherein the detection device uses ultrasound to detect the target particle of interest.

10. The system of claim 1 further comprising a series of flow paths for simultaneously examining multiple samples.

11. A method for selecting or enriching target particles of interest in a fluid sample, comprising: a flow path with an examination zone and a branching end for a rejected particle path and a collection path having a valve; a positioning device for positioning particles in the flow path such that, when the valve is open, the particles in the flow path will flow into the rejected particle path but will not flow into the return path; and a detection device for scanning particles within the scanning zone and in communication with the valve; providing a position assisted negative particle rejection (PANR) system having: when the detection device detects a target particle in the examination zone, the valve is closed, forcing the target particle into the collection path; directing said fluid sample through said flow path, examining and collecting said fluid sample; Recovering the contents in the recovery passage; directing the collected contents of the collection channel back into the flow path; and Repeated screening and collection until desired levels of selection and / or enrichment are achieved. The method according to any one of claims 1 to 4, 12. The method of claim 11, further comprising concentrating the recovered particles.

13. The method of claim 11, wherein the target particle is a cell.

14. The method of claim 11, wherein the target particle is a multicellular aggregate.

15. The method of claim 11, wherein the target particle is a small organism.

16. The method of claim 11, wherein the target particle is a nanoparticle.

17. The method of claim 11, wherein the target particle is a microsphere or a nanosphere.

18. The method of claim 11, wherein the apparatus is configured such that a portion of the fluid sample that does not contain particles flows into the collection path when the valve is opened.

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