Systems and methods of serial enrichment and dilution (SED) to sort target cells

Abstract

Provided are methods and apparatus for a novel serial enrichment and dilution (SED) process to sort target cells, including rare cells, from a mixture, allowing for the isolation of the target cells using serial dilution. The apparatus including a flow cell with a single flow path having an analysis zone between an inlet channel and a first outlet channel, a valve within the first outlet channel, and a second outlet channel attached to the first outlet channel, the valve opening one of the first or second outlet channel and closing the other of the first or second outlet channel when target cells are detected.

Description

Attorney Docket No. B359-0006PCT Filed: April 9, 2025 SYSTEMS AND METHODS OF SERIAL ENRICHMENT AND DILUTION (SED) TO SORT TARGET CELLS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 632,771, filed April 11, 2024, the contents of which are incorporated by reference herein in their entirety. FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to cell sorting and enrichment. BACKGROUND

[0003] Cell sorting and enrichment refers to the process of isolating and purifying a specific cell population from a heterogeneous mixture and has applications in many areas of biology and biomedicine (Robinson and Roederer 2015, Shields IV et al.2015). However, single cell sorting is too slow for many important applications (Piyasena et al., 2014, Shields et al., 2015, Zbrowski, et al., 2011, Brackenbury 2017). In an effort to increase sorting speed, mixtures may be pre- enriched prior to being sorted to increase the concentration of specific cell types in a sample.

[0004] Techniques that rapidly enrich a subset of cell types based on their physical properties (e.g. size, density, compressibility) are often used as the first step of sorting. However, this approach does not result in a high purity product as, with few exceptions, most biological cells are relatively similar in size, shape, compressibility, and density, as well as other physical parameters. Problems with current methods of separation are only made worse by the fact that cells within a specific population display significant size and shape variation, further impacting the ability of current enrichment processes to isolate specific cell populations. Thus, while it is possible to separate a few unique cell types from other types, most enrichment technologies that rely on intrinsic cell properties do not result in large gains in purity. Further, added enrichment steps increase the chance that rare cells will be lost while also increasing the complexity and cost of sorting (Sequist et al., 2009). SUMMARY OF THE DISCLOSURE

[0005] According to various embodiments, the present disclosure provides methods and apparatus for a serial enrichment and dilution (SED) process to sort rare target cells. Particular embodiments include a system for sorting and / or enriching one or more types of target particlesAttorney Docket No. B359-0006PCT Filed: April 9, 2025 in a fluid sample using a flow cytometer with a flow cell having a flow channel with a single flow path.

[0006] A heterogenous sample may be flowed through the flow cell and past an analysis zone located between an inlet channel and a first outlet channel of the flow cell. A detection device within the analysis zone interrogates the sample and identifies the presence or absence of target cells. The detection device may be any type of detection device including photodiodes, avalanche photodiodes, photomultiplier tubes, and silicon photomultipliers as well as other optical, imaging, ultrasound, or electrochemical methods. In some aspects, the target particles are attached to a detectable label. In other aspects, the target particles are identified based on other characteristics

[0007] When a detection device identifies target particles within the analysis zone, a signal is sent to a valve mechanism located within the first outlet channel, closing the first outlet channel and opening a second outlet channel attached to the first outlet channel (the sort position), diverting a portion of the sample through the second outlet channel. In some aspects, the valve may be positioned within the first outlet channel such that when the first outlet channel is open any second or secondary outlet channels are closed. That is, in an “open” position, the valve is positioned such that the first outlet channel is opened and the second outlet channel is closed. In a “closed” position (also referred to as a sort position), the valve closes the first outlet channel and opens the second outlet channel.

[0008] If no target cells are detected in the analysis zone, the valve remains in the open position, and that portion of the sample continues through the first outlet channel to a collection point. The process continues with the next portion of the sample, opening or closing the first outlet channel and one or more second outlet channels depending on the presence or absence of target cells.

[0009] Once a sample is processed, the portion of the sample collected from a second or secondary channel is diluted to the volume of the original sample and processed one or more additional times until the desired level of purity is achieved. With each processing cycle, the number of target cells in the sample relative to non-target cells increases.

[0010] To the accomplishment of the foregoing and related ends, certain illustrative aspects of the system are described herein in connection with the following description and the attached drawings. The summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of any subject matter described herein.Attorney Docket No. B359-0006PCT Filed: April 9, 2025 BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following figures, which form a part of this disclosure, are illustrative of described technology and are not meant to limit the scope of the claims in any manner.

[0012] FIGs.1A-1C depict an exemplary system and process for serial enrichment and dilution.

[0013] FIG.2 is an embodiment of a process for serial enrichment and dilution.

[0014] FIG.3 is an embodiment of a process for recovering a highly purified sample using the process of FIG.2.

[0015] FIG.4 is a graph showing target cell purity (%) as a function of time at different sample flow rates for a 1000 target cell / mL suspension.

[0016] FIG.5 is a graph showing target cell purity (%) as a function of time at different sample flow rates for a 1 target cell / mL suspension.

[0017] FIG. 6 is a flow chart of the steps and processing time for rare cell sorting using direct serial enrichment and dilution (SED).

[0018] FIG.7 is a flow chart of the steps and processing time for rare cell sorting using lysis serial enrichment and dilution (L-SED).

[0019] FIG. 8 is a flow chart of the steps and processing time for rare cell sorting using conventional magnetic enrichment followed by conventional flow cell sorting. DETAILED DESCRIPTION

[0020] The present disclosure provides methods and apparatus for a novel serial enrichment and dilution (SED) process to sort cells of interest (target cells), including rare target cells (i.e. cells present in a ratio of less than 1:1000 cells). For the purposes of the present disclosure, the term “sorting” is intended to mean the isolation of a very specific cell or particle type on a one by one basis based on its cellular function or particle characteristics, identification of which usually requires the detection of at least one biological or physical markers. “Enrichment” is defined as increasing the relative concentration of a given cell or particle type as compared to the starting mixture of cells or particles. For simplicity, while the present disclosure may refer to biological “cells,” it will be understood that the present disclosure is equally applicable to other particles in an aqueous fluid (e.g. microspheres, multicellular aggregates, small organisms, nanoparticles (including biological nanoparticles such as viruses and prions) etc.) Such targets may be referred to as target cells or target particles interchangeably.

[0021] Current enrichment processes including centrifugation, acoustophoresis, and filtration are based on the response of a cell to a force. However, as most biological cells are relatively similar in size, shape, compressibility, and density among other physical parameters, these approachesAttorney Docket No. B359-0006PCT Filed: April 9, 2025 do not lead to a high purity product and additional steps are generally needed to obtain isolation of the desired cell population. Further, current techniques such as fluorescent activated cell sorting (FACS), magnetic activated cell sorting (MACS), cell affinity chromatography, microfiltration, and immunoaffinity capture are not without their drawbacks. For example, MACS enriches a population of cells by binding a magnetic particle to the cell population via an antibody or similar molecule. However, there is potential cell damage due to strong magnetic fields and the labeling step may be insufficiently specific to extract the desired cell population.

[0022] For particles larger than single cells (i.e. >50 µm in diameter), the choice of sorting technology becomes even more restrictive. Large particle sorting technology is similar to FACS, but sorting is accomplished using a puff of air to displace a flowing jet in air stream (Chung, 2008; Pulak, 2006). Due to limitations with hydrodynamic focusing of larger particles and the relatively slow sort mechanism, FACS is limited to sorting about 100 particles per second at ˜200 mm diameter and is even slower for larger particles.

[0023] Conventional flow cytometry uses low flow rates on the order of 10 to 100 microliters per minute for sample delivery. This very low flow rate prevents large volume samples from being sorted effectively. Current pre-enrichment steps add complexity, require additional processing time, and lead to loss of rare cells—all of which are highly problematic for rare cell isolation from large volume samples.

[0024] The systems and methods described herein reduce the number of steps to achieve enrichment, providing a more efficient and less expensive process that isolates target cells with high yield and little target cell loss in less time than conventional cell sorting. Using serial dilutions and microfluidics, the systems and methods described herein capture target particles substantially free from non-target particles without additional processing.

[0025] FIGs. 1A-1C depict an embodiment of the SED process. In the SED process, a heterogenous suspension expected to contain one or more cells of interest is mixed with a label that binds to the cells of interest (target cells 102). “Label” or “labeled” as used herein refers to the addition of a detectable moiety to the cells for example, a radiolabel, fluorescent label, enzymatic label, chemiluminescent labeled, or a biotinyl group. Exemplary radioisotopes or radionuclides may include 3H, 14C, 15N, 35S, 90Y, 99Tc, mIn, 125I, 131I. Exemplary fluorescent labels may include rhodamine, lanthanide phosphors or FITC. Exemplary enzymatic labels may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase. Exemplary chemiluminescent labels include isoluminol, luminol and the dioxetanes. In some aspects multiple labels may be used. For example, labels may be bound to one or more markers on a target cell.Attorney Docket No. B359-0006PCT Filed: April 9, 2025 In some aspects different labels may be bound to different types of cells, allowing for identification and isolation of different types of target cells in a single sample.

[0026] In some aspects, the heterogenous suspension (also referred to herein as a mixture) may be pretreated prior to being mixed with the label. For example, the cells in the suspension that are not of interest may be lysed prior to combining the suspension with a label, decreasing the total number of cells that need to be sorted. Exemplary pretreatment includes the use of detergents, enzymes, surfactants, organic solvents, chelating agents, chaotropic agents, salts such as ammonium chloride, and the like.

[0027] As shown in FIG.1A, the suspension containing one or more labeled target cells 102 and non-targeted cells 104 is introduced into the flow cell of a flow cytometer on the left via an inlet 122. The suspension flows as shown by the arrows from left to right through an analysis region 108 at a set rate. Analysis / detection of the labeled particles may be optical, image based, ultrasound, spectroscopy, and the like using, for example, laser emitter 110. While a single analysis region is shown, in some aspects there may be multiple analysis regions. In embodiments, the approach can also include one or more detection systems with multiple interrogation points as long as the acquisition system can identify target cells 102 and generate an accurate sort signal. For example, a detection system may include the use of multiple lasers whether emitted from a single device or multiple devices or there may be primary and secondary detection systems such as a camera and other photodetectors including photomultipliers and photodiodes. In some aspects, the cells may be positioned prior to flowing through the analysis region 108 using for example, an acoustic standing wave, inertial focusing, dielectrophoretic focusing, and the like. In some aspects, hydrodynamic focusing may be used. An acoustic standing wave 106 is shown for exemplary purposes.

[0028] If no target cells 102 are detected in the analysis region 108, the suspension flows through a first outlet channel 114 and is collected for other use or disposal. If a target cell 102 is detected in the analysis region 108 as shown in FIG.1B, a signal 116 is sent to the valve mechanism 112 and the valve 120 opens the second outlet channel 118 and closes the first outlet channel 114 as shown in FIG. 1C. While a single second outlet channel is shown, there may be a plurality of second outlet channels which may be used to collect different types of target cells from a sample.

[0029] The valve 120 remains in the ‘sort’ position allowing access into the second outlet path until sufficient time passes to allow the identified target cell(s) to enter the sorted channel. The valve mechanism 112 then switches the valve 120 back to the ‘unsorted’ position, closing the second outlet channel 118, and allowing the portion of the mixed cells not containing target cells of interest to continue to flow through the first outlet channel 114. The process repeats until theAttorney Docket No. B359-0006PCT Filed: April 9, 2025 suspension is finished processing. While the target cells are still mixed with a portion of non- targeted cells 104, the proportion of target cells 102 relative to the number of non-targeted cells 104 increases, resulting in an enriched suspension at the end of a first cycle.

[0030] The portion of the suspension containing the target cells of interest collected from the second outlet channel 118 may then be diluted to the same volume as the original heterogenous mixture and reintroduced into the flow cell of the flow cytometer as shown in FIG.1A to further separate the target cells. The process may be repeated as many times as desired with each cycle increasing the purity of the collection. With each subsequent dilution and sorting, the amount of non-targeted cells decreases, increasing the proportion of target cells of interest in the sample collected from the second outlet channel 118.

[0031] In part to relieve the limits of volumetric delivery, BennuBio, Inc. (Albuquerque, NM, USA) developed a new flow cytometer (sold under the trademark Velocyt®) which can detect multiple colors of scattered or emitted fluorescent light. It can process up to 200,000 cells / second at an extremely high sample flow rate of 10 milliliters / minute. Notably, this system processes the sample in 10 parallel streams, which brings the possibility of using this instrument as the analytical starting point of a 10 analysis stream SED process, where 10 streams each processing at 400,000 cells / milliliter (or 4,000,000 cell / milliliter total) could be pursued. Using the serial enrichment and dilution process as described herein, the streams would be combined into a single outlet such as first outlet channel 114.

[0032] As shown in FIG.2 in process 200, a sample is flowed through the flow cytometer at 202. Each portion of the sample is subjected to analysis using, for example, analysis region 108 of the flow cell of FIGs. 1A-1C. If no target cells are identified at 204, the sample continues to flow through the flow cytometer, exiting thought the first outlet channel 114. If a target cell is identified at 204, a signal such as signal 116 is sent at 206 to move a valve such as valve 120 after time D to the sort position, opening the second outlet channel and closing the first outlet channel, and the portion of the sample containing the cells of interest is captured from the second outlet channel 118. The valve then closes the second outlet channel at 208, re-opening the first outlet channel and the remainder of the sample continues to flow through the cytometer at 202 until the entire sample is processed.

[0033] Time D is the time it will take a target cell to get from the analysis region such as analysis region 108, to the division between the first outlet channel 114 and the second outlet channel 118 as regulated by a valve mechanism such as valve mechanism 112. Time D can be calculated as shown in Equation 1, where F is the volumetric flow rate and Va-vis the volume between the analysis point and the valve.Attorney Docket No. B359-0006PCT Filed: April 9, 2025

[0034] If the minimum allowable a sort decision signal such as signal 116 and activate the valve suchas Dmin, the maximum volumetric flow rate (Fmax) is defined as shown in Equation 2. To determine usable flow rates calculated. Va-vconsists of the region of the flow cell of the flowregion (Vfcpl), and the capillary tubing of the first outlet channel 114 from the flow cell to the valve (Vcap) and is calculated as shown in Equation 3.

[0035] Vfcplcan be estimated from the analysis region to theend of the focusing flow cell as a 130 and width (Wfc) of the flow channel, Vfcplto be calculated as shown in Equation 4.

[0036] Vcap can be estimated by the length 128 of the capillaryto the valve (Lc) and its inner diameter (ID) 126 (Equation 5).

[0037] Combining Equationsallows for the development of reasonable estimates of Va-v.

[0038] Combiningestimate of the maximum flow rate for the system. For example, a= 0.75 mm, Lc = 50 mm, and Dminof 0.1 s, would have a maximum flow rate estimate of 0.289 milliliters per second or 17 milliliters per minute. This estimate suggests that the described system could support average laminar flow rates of 10 milliliters per minute if Dmin is 0.1 seconds.

[0039] As there is a delay between the identification of a target cell 102 within the analysis region 108 and the target cell reaching the division between the first outlet channel 114 and the secondAttorney Docket No. B359-0006PCT Filed: April 9, 2025 outlet channel 118, the timing of the opening of the valve can be calculated for different flow rates as shown in Equation 8. Assuming a flow rate above in which Lpl= 10 mm, Hfc= 0.4 mm, Wfcto be 0.578 seconds. That is, if a target cell such as target cell 102 is detected and a signal such as signal 116 is sent to a valve mechanism such as valve mechanism 112 to change the position of the valve 120 to open the second outlet channel 118 within 0.578 seconds, the target cell such as target cell 102 will be captured in the second outlet channel 118. In some aspects, the valve mechanism 112 may have a response time, that is the system must process data and produce a sort decision, send the decision to the valve mechanism and there is a time required for opening and closing the valve. For example, it may take 70 milliseconds to process the data and 30 milliseconds for the valve to open or close generating a final sort time of 100 milliseconds though other timing may also be used.

[0040] The volume that is diverted through the second outlet channel 118 at any given identification of a target cell such as target cell 102 is the total volumetric flow rate (F) multiplied by the time that it takes the valve to open and close, which is simply two times the response time of the valve (RT) (Equation 9). Assuming a flow rate of 3response time of 50 milliseconds, each diverted sort would contain 5 microliters of the suspension.

[0041] Using Equation 10, and the relevant diluted cell concentrations (C), when a target cell is detected and then sorted, the sorted volume of a blood sample would be 5 microliters and would contain 250 WBCs and 250,000 RBCs.

[0042] The total time (t) it would(V) of a 100 milliliter sample at a flow rate (F) of 3 milliliters per minute would be 33.3 min, or 2000 seconds (Equation 11).

[0043] The average occurrence rate of a given cell (l) in a sort volume is defined as the average number of cells in the sort volume (Equation 12). Using the Poisson distribution, thecell type is not present in a given sort volume is given in Equation 13.Attorney Docket No. B359-0006PCT Filed: April 9, 2025 Therefore, the probability (P+) is given by equation 14.

[0044] Using Equation dilution of whole blood, and assuming that everya simple spreadsheet to see the effect of SED in isolation of target cells at concentrations of 1 to 1000 cells per milliliter in whole blood. Considering a 100-fold dilution, the maximum probability that a target cell would be present in a sort value would be given by using Equation 14 and using a 100-fold dilution of 1000 cells and a sort volume of 5 microliters. The type l error in this situation is 0.05, which computes to about 4.8% chance of a sort volume not having a target cell. More importantly at these concentrations, the likelihood of two target cells being in the same sort volume is extremely low.

[0045] Therefore, the total sorted volume (Vsort) is given simply as the total number of target cells (NT) in the sample multiplied by the volume that is diverted during a sort (VD) (Equation 15). Notably, this gives a maximum for Vsort as coincidences in the sort volume would result in less sorts and a lower total volume. .

[0046] As shown in FIG. 3, 200 until the desired level of purity is obtained. For example, a sample is run through the sort process shown in FIG.2 at 302. The enriched sample collected from second outlet channel 118 as shown in FIG.1C may then be collected at 304, diluted to the original sample volume at 306, and run through the sort process 200 at 308 N additional times until the desired level of purity is obtained and the final sample containing the target cells is collected at 310. By diluting the collected sample at 306 to the original volume amount, the total cell concentration in the diluted enriched sample will be greatly reduced, allowing for improved purity of collection of the target cells with each pass through the sorting process.

[0047] The Exemplary Embodiments and Example(s) below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Attorney Docket No. B359-0006PCT Filed: April 9, 2025 Exemplary Embodiments.

[0048] 1. A system for sorting and / or enriching a target particle in a fluid sample, the system including: a flow channel with a single flow path having an analysis zone, wherein the analysis zone is between an inlet channel and a first outlet channel: a valve within the first outlet channel; a second outlet channel attached to the first outlet channel, the valve in the first outlet channel configured to close and open the second outlet channel; and a detection device that interrogates particles within the analysis zone, wherein the detection device is in communication with the valve; wherein detection of a target particle of interest within the analysis zone by the detection device results in the valve closing the first outlet channel and opening the second outlet channel, diverting the target particle and fluid in the channel adjacent to the target particle to flow into the second outlet channel; and wherein detection of no target particle within the interrogation zone by the detection device results in the valve closing the second outlet channel and opening the first outlet channel.

[0049] 2. The system of embodiment 1, wherein the valve is downstream of the analysis zone.

[0050] 3. The system of embodiments 1 or 2, wherein the valve closing the first outlet channel closes the second outlet channel when the first outlet channel is open.

[0051] 4. The system of any of embodiments 1 to 3, wherein the detection device is an optical detection device.

[0052] 5. The system of embodiment 4, wherein the optical detection device is configured to generate a laser beam through which the particles pass.

[0053] 6. The system of any of embodiments 1 to 3, wherein the detection device uses imaging to detect the particles of interest.

[0054] 7. The system of any of embodiments 1 to 3, wherein the detection device uses ultrasound to detect the particle of interest.

[0055] 8. The system of any of embodiments 1 to 7, wherein the system is configured to identify multiple types of target particles at a same time.

[0056] 9. The system of any of embodiments 1 to 8, wherein the system includes a plurality of detection devices.

[0057] 10. A method of isolating target particles within a heterogenous suspension including:Attorney Docket No. B359-0006PCT Filed: April 9, 2025 flowing a first volume of the heterogenous suspension containing at least one target particle through a flow channel having an analysis zone between an inlet channel and a first outlet channel; detecting within the flow channel, via a detection device, particles within the heterogenous suspension; flowing the first volume through the first outlet channel; closing a valve in the first outlet channel when a target particle is detected, wherein closing the valve blocks the first outlet channel and opens a second outlet channel; and opening the valve in the first outlet channel and closing the second outlet channel when no target particles are detected, wherein the valve closing the first outlet channel and opening the second outlet channel is the same valve.

[0058] 11. The method of embodiment 10 further including: collecting the heterogenous suspension containing at least one target particle from the second outlet channel; and diluting the collected heterogenous suspension to a second volume, wherein the first volume and the second volume are a same volume.

[0059] 12. The method of embodiment 10 or 11 further including: flowing the second volume through the flow channel; detecting, via a detection device, particles within a heterogenous mixture; flowing the second volume through the first outlet channel; closing a valve in the first outlet channel when a target particle is detected, wherein closing the valve blocks the first outlet channel and opens a second outlet channel; and opening the valve in the first outlet channel and closing the second outlet channel when no target particles are detected, wherein the valve closing the first outlet channel and opening the second outlet channel is the same valve.

[0060] 13. The method of embodiment 12, wherein the second volume includes a lower cell density than the first volume.

[0061] 14. The method of embodiments 12 or 13, wherein the second volume includes a higher concentration of target particles than the first volume.

[0062] 15. The method of any of embodiments 11 to 14, wherein the target particles are labeled.

[0063] 16. The method of any of embodiments 11 to 15, wherein the target particles are labeled with a plurality of labels.

[0064] 17. The method of embodiment 16, wherein each label binds a distinct epitope on a target particle.Attorney Docket No. B359-0006PCT Filed: April 9, 2025

[0065] 18. The method of any of embodiments 11 to 17, wherein the heterogenous suspension includes a plurality of target particle types.

[0066] 19. The method of any of embodiments 11 to 18, wherein each target particle type is labeled with a different label.

[0067] 20. The method of any of embodiments 10 to 19, wherein the flow channel has a single flow path. Example 1:

[0068] Current cell sorters (both droplet and microfluidic valve-based sorters) sort at ~25,000 cells / second and flow at ~20 microliters / minute, requiring nearly an hour to sort a 1 milliliter sample and about 8 hours to sort one 10 milliliter sample. In order to decrease processing time, front end concentration or magnetic enrichment steps are used to reduce the number of non- target cells and the sample volume being processed by a conventional flow cytometer. Such steps result in cell loss and are binary in their logic (i.e. they use the presence or absence of a single marker). Materials and Methods. Isolation of white blood cells (WBCs) from a blood sample.

[0069] A blood sample containing 100 target cells / milliliter, that is five billion total cells / milliliter (including both red blood cells (RBCs) and white blood cells (WBCs)) is combined with fluorescent tagged CD45 antibodies to selectively label WBCs, resulting in a labelled cell concentration of 5,000,000 cells / mL. The labeled sample is diluted 1000-fold to 5,000,000 cells / milliliter by adding 999 mL of PBS. The labeled sample is then flowed at 10 milliliters / minute through the flow cytometer, processing the 1 liter sample in 100 minutes.

[0070] Using a laser, the labeled WBCs are identified in an analysis zone of the flow cytometer. If no WBCs are identified, the sample is collected from a first outlet channel. If labeled WBCs are identified, a signal is sent to a valve in the first outlet channel and the first outlet channel is closed and a second outlet is opened, diverting the target cells into a second collection stream. After processing, ~400 milliliters of the blood sample are collected at a concentration of ~100 cells / milliliter (total) and a WBC concentration of 0.25 cells / milliliter.

[0071] The ~400 milliliters containing labeled WBCs is then diluted with PBS to a total volume of 1 liter. The sample is then flowed a second time at 10 milliliters / minute through the flow cytometer, processing the 1 liter sample in 100 minutes. Using a laser, the labeled WBCs are identified in an analysis zone of the flow cytometer. If no WBCs are identified, the sample is collected from the first outlet channel. If labeled WBCs are identified, a signal is sent to a valve and the first outlet channel is closed and a second outlet channel is opened, diverting the target cells into the secondAttorney Docket No. B359-0006PCT Filed: April 9, 2025 collection stream. After processing, ~400 milliliters of the mixture are collected at a concentration of ~100 cells / milliliter (total) and a WBC concentration of 0.25 cells / milliliter. Example 2:

[0072] Different flow rates impact the number of times a sample needs to be run through SED and amount of time needed to process a sample to a desired purity. Materials and Methods. Determination of flow rates relative to sample purity.

[0073] Five blood samples of 100mL with a starting target cell concentration of 1000 cell / mL is combined with fluorescently tagged CD45 antibodies. Each labeled sample is then flowed through the flow cytometer at 0.5ml / min, 1 ml / min, 3 ml / min, 5 ml / min and 10 ml / min respectively. Using a laser, the labeled WBCs are identified in an analysis zone of the flow cytometer. If no WBCs are identified, the sample is collected from a first outlet channel. If labeled WBCs are identified, a signal is sent to a valve and the first outlet channel is closed and a second outlet channel is opened, diverting the portion of the sample containing the labeled WBCS to the second outlet channel for collection. After processing the sample, the second outlet channel is flushed with 95 milliliters of cell media or buffer to collect the target cells resulting in a collection of a sample that has the original volume of 100 mL but has diluted the background cell concentration of RBCs and non-target WBCs.

[0074] The collected, diluted samples containing the WBCs are then flowed through the flow cytometer a second time at 0.5ml / min, 1 ml / min, 3 ml / min, 5 ml / min. and 10 ml / min respectively and the process is repeated ten times for each sample. As shown in FIG.4, a 3 or 5 millimeter flow would achieve purity in 3 to 4 hours. Example 3: Materials and Methods. Determination of flow rates relative to sample purity.

[0075] Five blood samples of 100mL with a starting target cell concentration of 1 cell / mL are combined with fluorescently tagged CD45 antibodies. Each labeled sample is then flowed at 0.5 ml / min, 1 ml / min, 3 ml / min, 5 ml / min. and 10 ml / min respectively through the flow cytometer. Using a laser, the labeled WBCs are identified in an analysis zone of the flow cytometer. If no WBCs are identified, the sample is collected from a first outlet. If labeled WBCs are identified, a signal is sent to a valve and the first outlet is closed and a second outlet is opened. After processing the sample, the second outlet is flushed with 95 milliliters of cell media or buffer to collect the target cells resulting in a collection of a sample that has the original volume of 100 milliliters but has diluted the background cell concentration of RBCs and non-target WBCs. The process is thenAttorney Docket No. B359-0006PCT Filed: April 9, 2025 repeated ten times for each sample. As shown in FIG.5, 3-5 mL / min sample flow rate would reach 100% target cell purity in <2 hours. Example 4:

[0076] Current sorters (both droplet and microfluidic valve-based sorters) sort at ~25,000 cells / second and flow at ~20 microliters / minute, it would take the greater part of an hour to sort a 1 milliliter sample and about 8 hours to sort one 10 milliliter sample. The SED process greatly reduces this time and can further accelerate processing by including an additional lysis step as shown in FIG.7. Materials and Methods.

[0077] Lysis-SED (L-SED). Ammonium chloride is added to a 1 mL heterogenous sample containing 100 target cells / milliliter, that is five billion total cells / milliliter (including both red blood cells (RBCs) and white blood cells (WBCs)). The mixture is incubated for 10 minutes and the WBCs are recovered. The suspension containing the WBCs is then combined with fluorescent tagged CD45 antibodies to selectively label the WBCs. The labeled sample is diluted with PBS to 100mL. The labeled sample is then flowed at 10 milliliters / minute through the flow cytometer. Using a laser, the labeled WBCs are identified in an analysis zone of the flow cytometer. If no WBCs are identified, the sample is collected from a first outlet channel. If labeled WBCs are identified, a signal is sent to a valve and the first outlet channel is closed and a second outlet is opened, diverting the portion of the sample containing the labeled WBCs into a second collection path. After processing, ~400 milliliters of the mixture are collected at a concentration of ~100 cells / milliliter (total) and a WBC concentration of 0.25 cells / milliliter.

[0078] The second outlet channel is then flushed with 95 milliliters of cell media or buffer to collect the target cells resulting in a collection of a sample that has the original volume of 100 milliliters with diluted background cell concentration of non-target WBCs. The process is repeated until the desired purity is achieved. Example 5: Comparison of SED to current flow sorter methods.

[0079] For the direct SED process 600 (FIG.6), the 1 milliliter sample is incubated with labelling reagents for 30 minutes at 602. The sample is then diluted to 1 liter and flowed through the flow cytometer a first time. In the flow cytometer, the labeled WBCs are identified using a laser in an analysis zone. If no WBCs are identified, the sample is collected from a first outlet channel. If labeled WBCs are identified, a signal is sent to a valve and the first outlet channel is closed andAttorney Docket No. B359-0006PCT Filed: April 9, 2025 a second outlet channel is opened. After processing the sample, the second outlet channel is flushed with 95 milliliters of cell media or buffer to collect the target cells resulting in a collection of a sample that has the original volume of 1 liter but has diluted the background cell concentration of RBCs and non-target WBCs. This process is repeated twice at 604 and the target cells are collected via acoustophoresis or centrifugation at 606 for a total processing time of 175 minutes. Thus, the SED work cycle would take about 3 hours in total but would not require RBC lysis or magnetic enrichment steps, which also require more processing and reagent costs.

[0080] Using the L-SED approach 700 (FIG. 7), ammonium chloride is added to a 1 mL blood sample containing 100 target cells / milliliter, that is five billion total cells / milliliter (including both red blood cells (RBCs) and white blood cells (WBCs)). The blood sample is incubated for 10 minutes and the WBCs are recovered at 702. The WBCs are then combined with fluorescent tagged CD45 antibodies to selectively label WBCs. The labeled sample is diluted with PBS to 100mL at 704. The labeled sample is then flowed at 10 milliliters / minute through the flow cytometer. Using a laser, the labeled WBCs are identified in an analysis zone of the flow cytometer. If no WBCs are identified, the sample is collected from a first outlet channel. If labeled WBCs are identified, a signal is sent to a valve and the first outlet channel is closed and a second outlet channel is opened, diverting the sample containing the target cells to a second collection point. After processing the sample, the second outlet is flushed with 999 milliliters of cell media or buffer to collect the target cells resulting in a collection of a sample that has the original volume of 1 liter but has diluted the background cell concentration of RBCs and non-target WBCs. This process is repeated twice at 706. The target cells are collected via acoustophoresis or centrifugation at 708 for a total processing time of 65 minutes for a 1mL sample.

[0081] In comparison, current cell sorter workflows 800 (FIG.8) likely start with RBC lysis at 802, which takes 10 minutes and a centrifugation step. This would be followed by magnetic enrichment of selected WBCs at 804. Magnetic enrichment would select for a single epitope using an antibody attached to a bead, which takes two steps. The first step 802, incubation of the cells with the magnetic beads, takes 30 minutes. The second step 804, harvesting of the cells labeled with magnetic beads, takes ~30 minutes and includes several wash steps. These harvested cells are resuspended in a volume that provides a workable cell concentration for flow analysis, which can vary, but for this purpose we will use 5 milliliters as a resuspension volume at 806. The resuspended cells are then labeled with fluorescent antibodies to additional markers of interest, which takes 30 minutes at 808. The labeled cells are then run through a conventional flow cytometry sorter at a rate of 20 microliters / minute (or less on many instruments) at 810. Therefore, at best this sorting step will take roughly 250 minutes. Thus, the typical approach will minimallyAttorney Docket No. B359-0006PCT Filed: April 9, 2025 take about 6 hours. Moreover, the typical approach takes several more steps than the SED approach. Thus, the presently described approaches are far simpler and can achieve at least a 2-fold faster rate than existing processes.

[0082] References

[0083] 1. Robinson, J. P. and M. Roederer, Flow cytometry strikes gold. Science, 2015. 350(6262): p.739-740.

[0084] 2. Shields IV, C. W., C. D. Reyes, and G. P. López, 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.

[0085] 3. Piyasena, M. E. and S. W. Graves, The intersection of flow cytometry with microfluidics and microfabrication. Lab on a Chip, 2014.14(6): p.1044-1059.

[0086] 4. Zborowski, M. and J. J. Chalmers, Rare cell separation and analysis by magnetic sorting.2011, ACS Publications.

[0087] 5. Brackenbury, W. J., High-Throughput, Low-Loss, Low-Cost and Label-Free Cell Separation using Electrophysiology Activated Cell Enrichment (EPACE). Proceedings of the National Academy of Sciences of the United States of America, 2017: p.1-6.

[0088] 6. Sequist, L. V., et al., The CTC-chip: an exciting new tool to detect circulating tumor cells in lung cancer patients. Journal of Thoracic Oncology, 2009.4(3): p.281-283.

[0089] 7. Chung, K. H., M. M. Crane, and H. Lu, Automated on-chip rapid microscopy, phenotyping and sorting of C. elegans. Nature Methods, 2008.5(7): p.637-643.

[0090] 8. Pulak, R., Techniques for analysis, sorting, and dispensing of C-elegans on the COPAS™ flow-sorting system, in Methods in Molecular Biology, K. Strange, Editor.2006, Humana Press Inc, 999 Riverview Dr, Ste.208, Totowa, N.J.07512-1165 USA. p.275-286. Closing Paragraphs

[0091] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients orAttorney Docket No. B359-0006PCT Filed: April 9, 2025 components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the amount and / or purity of the target cell collection.

[0092] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0093] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0094] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. NoAttorney Docket No. B359-0006PCT Filed: April 9, 2025 language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0095] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0096] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0097] Furthermore, numerous references have been made to patents, printed publications, journal articles, other written text, and web site content throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching(s), as of the filing date of the first application in the priority chain in which the specific reference was included. For instance, with regard to chemical compounds, nucleic acid, and amino acids sequences referenced herein that are available in a public database, the information in the database entry is incorporated herein by reference as of the date of an application in the priority chain in which the database identifier for that compound or sequence was first included in the text.

[0098] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0099] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in theAttorney Docket No. B359-0006PCT Filed: April 9, 2025 cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0100] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 11th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006), and / or A Dictionary of Chemistry, 8thEdition (Ed. J. Law & R. Rennie, Oxford University Press, 2020).

Claims

Attorney Docket No. B359-0006PCT Filed: April 9, 2025 LISTING OF CLAIMS What is claimed is:

1. A system for sorting and / or enriching a target particle in a fluid sample, the system comprising: a flow channel with a single flow path having an analysis zone, wherein the analysis zone is between an inlet channel and a first outlet channel: a valve within the first outlet channel; a second outlet channel attached to the first outlet channel, the valve in the first outlet channel configured to close and open the second outlet channel; and a detection device that interrogates particles within the analysis zone, wherein the detection device is in communication with the valve; wherein detection of a target particle of interest within the analysis zone by the detection device results in the valve closing the first outlet channel and opening the second outlet channel, diverting the target particle and fluid in the channel adjacent to the target particle to flow into the second outlet channel; and wherein detection of no target particle within the interrogation zone by the detection device results in the valve closing the second outlet channel and opening the first outlet channel.

2. The system of claim 1, wherein the valve is downstream of the analysis zone.

3. The system of claim 2, wherein the valve closing the first outlet channel closes the second outlet channel when the first outlet channel is open.

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

5. The system of claim 4, wherein the optical detection device is configured to generate a laser beam through which the particles pass.

6. The system of claim 1, wherein the detection device uses imaging to detect the particles of interest.

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

8. The system of claim 1, wherein the system is configured to identify multiple types of target particles at a same time.

9. The system of claim 1, wherein the system comprises a plurality of detection devices.

10. A method of isolating target particles within a heterogenous suspension comprising:Attorney Docket No. B359-0006PCT Filed: April 9, 2025 flowing a first volume of the heterogenous suspension containing at least one target particle through a flow channel having an analysis zone between an inlet channel and a first outlet channel; detecting within the flow channel, via a detection device, particles within the heterogenous suspension; flowing the first volume through the first outlet channel; closing a valve in the first outlet channel when a target particle is detected, wherein closing the valve blocks the first outlet channel and opens a second outlet channel; and opening the valve in the first outlet channel and closing the second outlet channel when no target particles are detected, wherein the valve closing the first outlet channel and opening the second outlet channel is the same valve.

11. The method of claim 10 further comprising: collecting the heterogenous suspension containing at least one target particle from the second outlet channel; and diluting the collected heterogenous suspension to a second volume, wherein the first volume and the second volume are a same volume.

12. The method of claim 11 further comprising: flowing the second volume through the flow channel; detecting, via a detection device, particles within a heterogenous mixture; flowing the second volume through the first outlet channel; closing a valve in the first outlet channel when a target particle is detected, wherein closing the valve blocks the first outlet channel and opens a second outlet channel; and opening the valve in the first outlet channel and closing the second outlet channel when no target particles are detected, wherein the valve closing the first outlet channel and opening the second outlet channel is the same valve.

13. The method of claim 12, wherein the second volume comprises a lower cell density than the first volume.

14. The method of claim 12, wherein the second volume comprises a higher concentration of target particles than the first volume.

15. The method of claim 10, wherein the target particles are labeled.

16. The method of claim 15, wherein the target particles are labeled with a plurality of labels.

17. The method of claim 16, wherein each label binds a distinct epitope on a target particle.

18. The method of claim 10, wherein the heterogenous suspension comprises a plurality of target particle types.Attorney Docket No. B359-0006PCT Filed: April 9, 2025 19. The method of claim 16, wherein each target particle type is labeled with a different label.

20. The method of claim 10, wherein the flow channel has a single flow path.

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