Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties

Definitions

• the present invention relates in general to fluid analysis systems, such as flow cytometers, and is particularly directed to a process for analyzing a fluid, such as one containing human blood cells, for the presence of prescribed constituents, such as preselected types of blood cells, and then sorting successively generated fluid droplets to a selected one of a plurality of collection stores based upon the constituency of a selected plurality of contiguous droplet precursor regions.
• a fluid such as one containing human blood cells
• prescribed constituents such as preselected types of blood cells
• Flow cytometers are commonly employed in the medical industry to analyze particles in a patient's body fluid (e.g., blood cells) as an adjunct to the diagnosis and treatment of disease.
• a patient's body fluid e.g., blood cells
• such instruments may be used to sort and collect healthy blood cells (stencils) from a quantity of blood that has been removed from a patient's bone marrow prior to chemotherapy. Once a chemotherapy treatment session is completed, a collected quantity of these cells is then reinjected back into the patient, to facilitate migration and healthy blood cell reproduction.
• particles 10 to be analyzed are analyzed by injecting the particles into a (pressurized) continuous or uninterrupted carrier fluid (e.g., saline) 14, and directing the carrier fluid along a channel 15 that passes through the output beam 16 emitted by an optical illumination subsystem, such as one or more lasers 18.
• an optical illumination subsystem such as one or more lasers 18.
• Located optically in the path of the laser output beam 16 after its being intercepted by the carrier fluid passing therethrough are one or more photodetectors of a photodetector subsystem 21.
• This photodetector subsystem 21 is positioned to receive light as modulated by the contents of (particles/cells within) the fluid in the channel 15, including that reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell.
• the fluid flow channel through the cytometer is configured and sized to pass the particles or cells only one cell at the time through an intersection location 19 with the laser's output beam 16.
• each modulation signal can be associated with a respective cell in the fluid carrier stream.
• the photodetector output signal may be used to control the sorting of a droplet of the carrier fluid containing that cell (by means of a downstream droplet sorter 30), once that droplet is formed by into a stream of droplets 23 by means of an acoustically (e.g., piezoelectric) driven droplet generator 25.
• the photodetector output is typically digitized and then analyzed by a cell type mapping or identification algorithm executed by an associated supervisory control processor. Based upon this analysis, the control processor instructs the droplet sorter to sort or abort the droplet.
• the sort delay is the period of time that elapses between the instant in time at which the photodetector generates an output signal for that cell as it is illuminated by the laser beam at intersection location 19 and the time at which that portion of the fluid stream containing that cell breaks off into a droplet at some downstream location 29.
• the sort delay may be defined means of by a delay timer, such as a counter driven by a high speed sort control clock, so that the output of the counter indicates the time difference between the time that the portion of region of the carrier fluid containing the cell of interest is irradiated by the laser beam to the time when a droplet containing the cell of interest eventually separates from the carrier fluid stream.
• the individual droplets 23 produced by the droplet generator 25 do not form immediately at the droplet generator's exit port 27, but rather break off naturally and in random fashion at location 29 downstream of the exit port 27.
• the point in space 29 downstream of the exit port 27 at which the droplets individually form may be adjusted by varying the parameters of the drive signal to the piezoelectric transducer of the droplet generator 25, and may be defined so as to cause the droplets 23 to become synchronized with the frequency of the piezo vibration of the droplet generator 25.
• the acoustic drive frequency applied to the droplet generator 25 may be on the order of from four to one hundred Khz, at a fluid pressure on the order of from three to seventy psi.
• the droplet sorter 30 may be controllably sorted by means of droplet sorter 30 into a collection container 41, or allowed to pass unsorted along a main travel path 24 into an aborted or discarded waste container 43.
• the droplet sorter's electrostatic charging collar 31 may comprise a metallic ring surrounding that point in the droplet stream 22 where the individual droplets 23 separate from the fluid stream, and is typically several droplets in length. It is positioned vertically downstream of the exit port 27 of the droplet generator 25, and upstream of an associated set of electrostatic (opposite polarity, high voltage) deflection plates 33 and 35 between which the stream of charged droplets 23 pass as they travel downwardly towards the collection and waste containers.
• a prescribed charging voltage is selectively applied via deflection control circuitry 52 to the charging collar 31 at a time determined by the sort delay, thereby charging that segment of the cell-containing fluid stream, so that any droplet breaking off from the stream at that point and containing the cell of interest will carry the charge induced by the collar. Then, as an individual charged droplet carrying this charge (one of which is shown at 23C) passes between the two opposite polarity high voltage deflection plates 33 and 35, it is attracted to the plate with the opposite charge, while being simultaneously repelled by the plate with the same or like charge.
• this electrostatic steering action directs the charged droplet 23C along a path 26 to one side of the travel path 24 of the main stream, and into the collection container 41 placed on the side of the path of the main stream of droplets.
• the location of the point 29 where an individual droplet 23 forms or breaks off from the continuous fluid stream 22 is critical to accurate sorting of the droplets, since only a droplet that breaks off from the stream at the time of the applied sort charge will be deflected by the deflection plates, and subsequently collected in the target sorting container.
• sort delay between the time at which the photodetector subsystem 21 generates an output signal for that cell and the time at which a droplet 23 containing that cell breaks off from the fluid stream.
• the output signal from the photodetector is digitized and then analyzed-processed by a droplet sort routine executed by the system's control processor (controlled by or installed within workstation 50) to determine whether the droplet containing the cell of interest is to be sorted into the collection container 41 or aborted into the waste container 43.
• a preliminary calibration cycle is conducted to accurately locate the droplet formation point.
• this may be accomplished by manually placing the droplet formation point 29 at a predetermined distance from the laser intersection point 19, using a microscope objective, or a video system, to observe the fluid stream 22.
• a microscope objective or a video system
• the droplets 23 formed from the fluid stream 22 will appear to be stationary.
• the droplet formation point 29 can be moved closer or farther away from the laser intersection point 19.
• the system operator can then adjust the point at which the droplets first form to a reference or positioning mark.
• the operator inputs to the sorting system a sort delay time that has been determined on the basis of previous experimentation, so as to place the system within several droplets of the actual sort delay time.
• the operator sets up and runs a calibration sort operation, using test beads, which mimic biological cells in terms of size.
• the beads are sorted onto a slide, and the slide is observed with a microscope in order to determine if the number of beads on the slide coincides with the number of beads the system reported as having sorted. If the numbers do not coincide, then the system is adjusted by changing the sort delay time, or by moving the droplet formation point by adjusting the acoustic drive signal. This operation is iteratively repeated as necessary until the beads counts are correct. With the system thus initially calibrated, it may then be monitored visually for drift, with the operator observing the fluid stream and droplets for movement. To verify that the sort parameters remain the same, the slide and bead analysis sequence described above may be repeated.
• the particle (e.g., blood cell) processing rate of a flow cytometer is often limited to a relatively slow data rate (e.g., a range on the order of from ten to thirty thousand cells per second), then for commonplace yield rates of desired cells on the order of only five percent or less, the time required to collect or harvest a highly purified quantity of cells (e.g., on the order of a million or more), may be as long as six hours.
• a relatively slow data rate e.g., a range on the order of from ten to thirty thousand cells per second
• a new and improved flow cytometer sorting mechanism that is readily executable by the system's supervisory computer for controllably sorting successively generated fluid droplets to a selected one of a plurality of collection stores, the reclaimable contents of which have a useful cell density that is elevated considerably compared with that of the contents of an original storage container, such as that containing a centrifuged blood sample.
• the sorting routine of the invention is operative to sort a respective
• this multi-region sorting window scheme is particularly useful in reducing the time required to harvest a highly purified quantity of a particular type of cell.
• the droplet is sorted into an auxiliary 'enrichment' container, whose contents may then be reclaimed for resorting.
• the instrumentation architecture of a flow cytometer system employing multiple reclaimable collection stores in accordance with the present invention includes essentially the same components of the cytometer of Figure 1 , described above, but with droplets being sortable to any of a plurality of particle reclamation containers that are arranged on opposite sides of the main droplet travel path, or unsorted directly to a waste container.
• reclamation container is meant a sorted droplet collection container or store, from which a quantity of extremely pure sorted droplets may be reclaimed for further use (such as reinjection back into the patient, as in the case of chemotherapy treatment, described above), or from which a quantity of less than extremely pure, but having a considerably higher density of desired particles/cells than that of the original source may be retrieved for reprocessing through the cytometer sorting mechanism of the invention.
• the sort control routine executed by the system workstation is operative to sort a respective fluid droplet based upon the contents of a plurality of contiguous droplet precursor regions within that portion of the fluid stream from which the droplet of interest will be formed, and also in adjacent portions of the fluid stream from which droplets on either side of the droplet of interest will be formed.
• droplet precursor region is meant one of a plurality of sequentially contiguous regions within the particle/cell carrier fluid that will eventually be contained within a respective droplet that has broken off from the continuous fluid stream.
• the term 'precursor' reflects the fact that within the fluid stream at a location upstream of the droplet separation location, in particular within that portion of the carrier fluid illuminated by the laser output beam, the fluid stream may considered to be comprised of a sequence of contiguous regions that will eventually become, but are not yet formed into an actual droplet.
• the resolution to which the fluid within a respective droplet may be subdivided into a plurality of such droplet precursor regions may be established in accordance with the frequency of the clock signal, by which the sort delay between the laser beam intersection location and the downstream droplet formation location is measured, such as that used to clock a sort delay counter, described above.
• That portion of the continuous fluid flow stream, that will eventually break off from the stream and become a target droplet that is sorted into one of the plurality of reclamation containers or aborted, will comprise an interior-most or middle sequence of four droplet precursor regions within an overall sequence of twelve droplet precursor regions. Of the remaining eight regions, the first four are associated with an immediately adjacent downstream droplet and the last four are associated with an immediately adjacent upstream droplet.
• sorting modes may be executed by defining sorting criteria windows to determine whether the center or second (target) droplet in the sequence of three droplets is to be sorted or aborted and, if sorted, how it is to be sorted.
• the sorting modes may include the following modes.
• a maximum width, non-abort (abort off) mode has a sort window that contains all of the droplet precursor regions of the target droplet and each of its immediately adjacent droplets.
• This sorting mode is intended to provide the highest yield of desired cells, but also allows particles other than the desired to be collected, so that the purity of the desired cells can be expected to be less than one hundred percent.
• this first sorting mode if at least one desired cell/particle of interest is detected anywhere within the sort window (containing all twelve precursor regions for the three droplets), the target droplet is sorted to a reclamation container.
• a multi-sort mode may be used to selectively sort multiple types of cells to respective reclamation containers, or selectively sort the same type of cell to different reclamation containers, based upon the cell purity within the sort window.
• the target droplet When sorting multiple types of cells, the target droplet will be sorted to that one of the reclamation containers that has been predetermined to collect the type of cell of interest if the following conditions are satisfied.
• at least one of the different types of cells/particles of interest must be found anywhere within a prescribed multi-precursor region sort window, such as one containing all regions of the target droplet, and an immediately contiguous region of the adjacent downstream droplet, and the immediately contiguous region of the adjacent upstream droplet.
• a prescribed multi-precursor region sort window such as one containing all regions of the target droplet, and an immediately contiguous region of the adjacent downstream droplet, and the immediately contiguous region of the adjacent upstream droplet.
• This mode is designed to sort the target droplet containing only a given type of particle to a high purity container, but otherwise sorting the target droplet to an auxiliary enrichment container, rather than aborting the target droplet. No droplets are aborted.
• the target droplet will be sorted right to the enrichment container, the contents of which may be re-centrifuged and then processed again through the cytometer system of the invention.
• the target droplet In order to sort the target droplet to a high purity container, two requirements must be fulfilled. First, there must be at least one prescribed type of cell/particle within the sort window (e.g., a multi-region window containing regions of the target droplet and those of adjacent downstream and upstream droplets). Secondly, there must be no other type of cell or event within the sort window. Only if both of these requirements are satisfied will the target droplet be sorted to the high purity reclamation container. For all other cases, the target droplet will be sorted to the enrichment container.
• the sort window e.g., a multi-region window containing regions of the target droplet and those of adjacent downstream and upstream droplets.
• the multi-region sorting window scheme is particularly useful in reducing the time required to harvest a highly purified quantity of a particular type of cell.
• target droplets containing only desired cells sorted to a highly purified collection store but should an undesired cell be detected within a predefined proximity of a desired cell, rather than being aborted and therefore lost, the target droplet is sorted into the enrichment container, whose contents may then be reclaimed for resorting.
• all droplets are sorted to one of a plurality of reclamation containers, no cells are lost, as in conventional proximity based aborting decisions.
• the total harvest time can be significantly reduced.
• An additional abort-on mode may be used to achieve a very high purity of desired cells by the use of a relatively narrow inclusion window.
• This mode employs a first, relatively narrow sorting window for desired cells only, that is bounded by a second, relatively wide exclusion window, in which the presence of any particles/cells (including the desired type of particle) will cause the target droplet to be aborted.
• the width of the sorting window may consist of less than or all of the droplet precursor regions of the target droplet.
• Figure 1 diagrammatically illustrates the general instrumentation architecture of a flow cytometer
• Figure 2 diagrammatically illustrates the general instrumentation architecture of a flow cytometer system employing multiple reclaimable collection stores in accordance with the present invention
• Figure 3 diagrammatically illustrates a plurality of droplet precursor regions within the carrier fluid that will eventually be contained within a respective target droplet broken off from the continuous fluid stream;
• Figure 4 shows a set of four successively contiguous droplet precursor regions
• Figure 5 diagrammatically illustrates the manner in which various modes of the sort control routine of the present invention controllably sort a respective target droplet based upon the contents of a plurality of contiguous droplet precursor regions for the target droplet and droplet precursor regions for droplets on either side of the target droplet.
• FIG. 2 diagrammatically illustrates the instrumentation architecture of a flow cytometer system employing multiple reclaimable collection stores in accordance with the present invention.
• the system comprises essentially the same components as shown in Figure 1 , described above, but with droplets being sortable to any of a plurality of particle reclamation containers 61 and 62 arranged on opposite sides of the main droplet travel path 24, or being unsorted directly to a waste or abort container 62.
• reclamation container is meant a sorted droplet collection container or store, from which a quantity of extremely pure sorted droplets may be reclaimed for further use (such as reinjection back into the patient, as in the case of chemotherapy treatment, described above), or from which a quantity of less than extremely pure, but having a considerably higher density of desired particles/cells than that of the original container 12, may be retrieved for reprocessing through the cytometer sorting mechanism of the invention.
• the sort control routine executed by the system workstation 50 is operative to sort a respective fluid droplet based upon the contents of a plurality of contiguous droplet precursor regions within that portion of the fluid stream from which the droplet of interest will be formed, and also in adjacent portions of the fluid stream from which droplets on either side of the droplet of interest will be formed.
• droplet precursor region is meant one of a plurality of sequentially contiguous
• the term 'precursor' is employed to reflect the fact that within the fluid stream 22 at a location upstream of the droplet separation location 29, including within that portion of the carrier fluid intersected by the laser output beam 16 at location 19, the fluid stream 22 may considered to be comprised of a sequence of contiguous regions that will 'eventually become, but are not yet formed into' an actual droplet -which breaks off from the uninterrupted fluid stream at droplet separation location 29.
• the resolution to which the fluid within a respective droplet 23 may be subdivided into a plurality N of such droplet precursor regions may be established in accordance with the frequency of the clock signal, by which the sort delay or fluid travel time between laser beam intersection location 19 and droplet formation location 29 is measured, such as that used to clock a sort delay counter, as described above.
• Sorting criteria windows employed by respective sorting modes MO - M8, to be described individually below, to determine whether the center or second (target) droplet 23-2 in the sequence of three droplets 23-1 , 23-2 and 23-3 is to be sorted or aborted and, if sorted, how it is to be sorted.
• This first sorting mode is intended to provide the highest yield of desired cells, but also allows particles other than the desired to be collected, so that the purity of the desired cells may be less than one hundred percent.
• a desired cell/particle of interest (labeled throughout Figure 5 as a 'good guy') is found anywhere within a maximum sized sort window - one containing all twelve precursor regions 22-1... 22-12, and regardless of the presence of an event/anomaly that might otherwise cause the droplet to be aborted (abort is turned off)
• a decision is made to sort the target droplet 22-2 to a selected one of the reclamation containers, such as the container 61 shown at the 'left' side of the droplet travel path 24.
• this sort decision is labeled as 'Priority SORT LEFT'. It should be noted that in this mode, in order to be sorted (left), the target droplet 23-2 need not actually contain a cell of interest. All that is required is that at least one of the twelve precursor regions 22-1... 22-12 contain at least one desired cell.
• This second sorting mode (and also the third and fourth sorting modes M2 and M3, respectively) is intended to sort either of two types of particles - one being sorted left to container 61 at the 'left' side of the droplet travel path 24, and the other being sorted right to container 62 at the 'right' side of the droplet travel path 24.
• the target droplet 23-2 will be sorted to that one of the reclamation containers that has been predetermined to collect the type of cell of interest only if the conditions set forth below are satisfied. Otherwise, the target droplet 23-2 will be aborted.
• At least one of either of the two prescribed cell/particles of interest must be found anywhere within a six precursor region sort window containing all four regions 22-5...22-8 of the target droplet 23-2, immediately contiguous region 22-4 of adjacent downstream droplet 23-1 , and immediately contiguous region 22-9 of upstream droplet 23-3.
• the fluid sample to be processed may contain two different types of blood cells that have been stained with red and green fluorescent dye antibodies, and which are to be collected in containers 61 and 62, respectively.
• the target droplet 23-2 will be sorted right to the green cell container 62. Otherwise, the target droplet will be aborted.
• the target droplet 23-2 will be sorted left to the red cell container 61. Otherwise, the target droplet 23-2 will be aborted.
• the target droplet 23-2 need not actually contain a cell of interest to be sorted either right or left. All that is required is that at least one of the six precursor regions 22-4...22-9 contain at least one desired cell, and no bad cell.
• this third sorting mode sorts either of two types of particles - one sorted left to container 61 at the 'left' side of the droplet travel path 24, and the other sorted right to container 62 at the 'right' side of the droplet travel path 24.
• Mode M2 is identical to mode M1 except that the sort window is expanded to cover a fifty percent overlap into the precursor regions of the adjacent droplets 23-1 and 23-3. Namely, in this third sorting mode, if either of two prescribed (good guy) cell/particles of interest is found anywhere within an eight precursor region window containing all four regions 22-5...
• the fourth sorting mode M3 is identical to modes M1 and M2 except that the sort window is expanded to cover a full one hundred percent overlap into all of the precursor regions of the adjacent droplets 23-1 and 23-3. Namely, in this fourth sorting mode, if at least one of either of two prescribed (good guy) cell/particles of interest is found anywhere within a twelve precursor region window containing all four regions 22-1 ... 22-4 of the first droplet 23-1 , all four regions 22-5 ... 22-8 of the target droplet 23-2, and all four regions 22-9 ... 22-12 of the third droplet 23-3, and there is no other detected type of cell or event within the twelve region window, then a decision is made to sort the target droplet 23-2 to that one of the reclamation
• each of these modes is a dual sort mode for a given type of cell. Dual sort provides a high purity quantity of the desired cell in one reclamation container, and also provides a secondary container of 'good guy' cells, that may be reclaimed for further sorting.
• the target droplet 23-2 will be sorted (right) to the other reclamation container 62, which effectively serves as an enrichment container. It may also be noted that in each of modes M4-M6, the target droplet 23-2 need not actually contain a cell of interest to be sorted left to the high purity container. All that is required is that at least one of the six precursor regions 22-4 ... 22-9 contain a desired cell and no bad cell.
• the sixth sorting mode is identical to the fifth sorting mode, described above, and is designed to 'sort left' only a given type of particle to the high purity container 61 , but otherwise sort the target droplet to the enrichment container 62.
• the eight precursor regions of the sorting window of mode M5 are all four regions 22-5 ... 22-8 of the target droplet 23-2, the two immediately contiguous regions 22-3 and 22-4 of adjacent downstream droplet 23-1, and the two immediately contiguous regions 22-9 and 22-10 of upstream droplet 23-3.
• Mode M6 is identical to modes M4 and M5 except that the sort window is expanded to cover a full one hundred percent overlap into all of the precursor regions of the adjacent droplets 23-1 and 23-3.
• the multi-region sorting window scheme of modes M4 - M6 is particularly useful in reducing the time required to harvest a highly purified quantity of a particular type of cell. Not only are target droplets containing only desired cells sorted to a highly purified collection store, but should an undesired cell be detected within a predefined proximity of a desired cell, rather than being simply aborted, the target droplet is sorted into an auxiliary 'enrichment' container, whose contents may then be
• the sorted desired cell only population can be expected to be reduced by some percent (e.g., only fifty percent), resulting in an actual high purity yield rate of 75 cells per second.
• the corresponding yield rate is 75 cells/sec.
• the enrichment container will accumulate a total quantity of 500K desired cells in 500K/7500 or 66.66 seconds (1.1 hours), namely within the time (1.85 hours) required for the high purity container to accumulate 500K cells.
• Modes M7 and M8 are intended to provide a very high purity of desired cells by the use of relatively narrow inclusion windows.
• Each of these two modes employs a first, relatively narrow sorting inclusion window for desired cells only, that is bounded by a relatively wide exclusion window, in which the presence of any particles/cells will abort the target droplet.
• Mode M7 is intended to provide a very high purity of desired cells. For this purpose, it employs a first, relatively narrow sorting inclusion window for desired cells only, that is bounded by a relatively wide exclusion window in
• this eighth sorting mode in order to keep or sort the target droplet 23-2 to a reclamation container, at least one desired cell/particle of interest ('good guy') and no other types of particles or events must be found within a relatively narrow sorting inclusion window that consists of the middle two precursor regions 22-6 and 22-7 for the target droplet 23-2. Secondly, the remaining precursor regions 22-1 ... 22-5 and 22-8 ... 22-12 must be empty. Otherwise the target droplet is aborted.
• Mode M8 is substantially the same as mode M7, except that the sorting inclusion window encompasses all of the precursor regions associated with the target droplet, and the exclusion window contains all of the precursor regions for the two adjacent droplets.
• this ninth sorting mode in order to keep or sort the target droplet 23-2 to a reclamation container, at least one desired cell/particle of interest ('good guy') and no other types of particles or events must be found within a sorting inclusion window that consists of all of the precursor regions 22-5 ... 22-8 for the target droplet 23-2.
• the precursor regions 22-1 ... 22-4 for the adjacent downstream droplet 23-1 and the precursor regions 22-9 ... 22-12 for the adjacent upstream droplet 23-3 must be empty. Otherwise the target droplet 23-2 is aborted.
• drawbacks of conventional flow cytometer sorting schemes are effectively remedied by the flow cytometer sorting mechanism of the invention, that is operative to controllably sort successively generated fluid droplets in accordance with the contents of a plurality of contiguous precursor regions of the carrier fluid which are contained within the target droplet of interest and precursor regions for droplets on either side of the target droplet.
• This multi-region sorting window scheme is particulariy useful in reducing the time required to harvest a highly purified quantity of a particular type of cell, as not only are droplets containing only desired cells sorted to a highly purified collection store, but should an undesired cell be detected within a predefined proximity of a
• the droplet is sorted into an auxiliary enrichment container, whose contents may then be reclaimed for resorting. Since all droplets are sorted to one of a plurality of reclamation containers, no detected/processed cells are lost, as in conventional proximity based aborting decisions. As a consequence, through dual collection and resorting of the contents of the enrichment container the total harvest time can be significantly reduced.

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