(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`( 43) International Publication Date
`14 October 2004 (14.10.2004)
`
`PCT
`
`(10) International Publication Number
`WO 2004/088283 A2
`
`(51) International Patent Classification7:
`
`G01N 15/00
`
`(21) International Application Number:
`PCT/US2004/009646
`
`(22) International Filing Date: 29 March 2004 (29.03.2004)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/458,607
`60/458,731
`
`28 March 2003 (28.03.2003) US
`28 March 2003 (28.03.2003) US
`
`(71) Applicant (for all designated States except US): MON(cid:173)
`SANTO TECHNOLOGY LLC [US/US]; 800 North
`Lindbergh Boulevard, St. Louis, Missouri 63102 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): DURACK, Gary
`[US/US]; 2505 Appaloosa Lane, Mahomet, IL 61853
`(US). HATCHER, Jeremy, T. [US/US]; 509 East Shurts
`
`Street, Urbana, IL 61801 (US). WESTFALL, Lon, A.
`[US/US]; 1007 Timber Drive, Mahomet, IL 61853 (US).
`HELBLING, David, R. [US/US]; 904 South Fawn
`Drive, Mahomet, IL 61853 (US). WALLACE, Jeffrey,
`D. [US/US]; 666 Autumn Fields Lane, Rantoul, IL 61866
`(US). VANDRE, Gary, P. [US/US]; 105 Sharon Drive,
`Mahomet, IL 61853 (US). DIDION, Bradley [US/US];
`220 Hickory Hollow, Washington, MO 63090 (US).
`NAYAK, Niraj, V. [US/US]; 504 Avenue G, Apt. #24,
`Redondon Beach, CA 90277 (US). ANZAR, Muhammad
`[CA/US]; 14506 Tienda Drive, Chesterfield, MO 63017
`(US). LUDWIG, Cindy, L. [US/US]; 1412 Dautel Lane,
`St. Louis, MO 63146 (US). GRAHAM, Jeffrey, A.
`[US/US]; 49 Picardy Hill Drive, Chesterfield, MO 63017
`(US). CROWLEY, Kathleen, S. [US/US]; 315 Carmel
`Road, Webster Groves, MO 63119 (US).
`
`(74) Agents: GODAR, Michael, E. eta!.; Senniger, Powers,
`Leavitt & Roedel, #1 Metropolitan Square, 16th Floor, St.
`Louis, Missouri 63102 (US).
`
`[Continued on next page}
`
`(54) Title: APPARATUS, METHODS AND PROCESSES FOR SORTING PARTICLES AND FOR PROVIDING SEX-SORTED
`ANIMAL SPERM
`
`(57) Abstract: Apparatus and methods for analyzing par(cid:173)
`ticles, including apparatus and methods for a sperm sorting
`process including: collecting sperm from an animal (30);
`selecting staining conditions ( 47 A); staining the sperm with
`DNA selective fluorescent dye ( 48); sorting the sperm cells
`according to sex chromosome content (55); and cryopre(cid:173)
`serving a population of sorted sperm (61) until used for ar(cid:173)
`tificial insemination. One embodiment includes apparatus
`(1001) and methods for using a plurality of flow cytometry
`units (9) sharing an integrated platform to sort sperm cells.
`In one embodiment, flow cytometric sorting includes use
`of the following apparatus and methods: an orienting noz(cid:173)
`zle having a baffle (101); an epi-illumination optics sys(cid:173)
`tem (109); slit scanning of localized DNA regions within
`cell nuclei (225); digital signal processing, including syn(cid:173)
`chronous sampling of analog output signals (701), pulse
`waveform ( 497) feature extraction of an approximation of a
`first order derivative of a pulse waveform ( 497) at a point of
`the pulse, any of various sort strategies; and an automated
`sort calibration system (4201). In one embodiment, digital
`signal processing includes sampling analog output signals
`(701) at times relative to emission of pulses from an illumi(cid:173)
`nation laser. Other embodiments are substantially different
`from the foregoing, including embodiments directed to in(cid:173)
`dividual steps or systems that can be used for any of various
`applications involving particle analysis.
`
`iiiiiiii
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`-iiiiiiii
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`---
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`!!!!!!!!
`iiiiiiii
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`iiiiiiii ----
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`649
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`3
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`

`

`WO 2004/088283 A2
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, Fl,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
`MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
`PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
`zw.
`
`Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), Euro(cid:173)
`pean (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl, FR,
`GB, GR, HU, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI, SK,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`

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`APPARATUS, METHODS AND PROCESSES FOR SORTING PARTICLES AND FOR
`
`PROVIDING SEX-SORTED ANIMAL SPERM
`
`Background of the Invention
`
`5
`
`This invention relates generally to apparatus and methods for animal semen collection,
`
`and more particularly to apparatus and methods using various techniques, including flow
`
`cytometry, to yield sperm populations that are enriched with sperm cells having one or more
`
`desired characteristics, such as viable populations of sperm cells sorted according to DNA
`
`characteristics for use by the animal production industry to preselect the sex of animal offspring.
`
`10
`
`The fertilization of animals by artificial insemination (AI) and embryo transplant following
`
`in vitro fertilization is an established practice. In the livestock production industry, the ability to
`
`influence the reproductive outcome toward offspring having one or more desired characteristics
`
`has ~bvious advantages. By way of example, there would be an economic benefit in the dairy
`
`industry to preselect offspring in favor of the female sex to ensure the production of dairy cows.
`
`15
`
`Efforts have been made toward achieving this goal by using flow cytometry to sort X andY
`sperm cells, as evidenced by the disclosures in US Patents Nos. 6,357,307 (Buchanan, et al.),
`5,985,216 (Rens, et al.), and 5,135,759 (Johnson). However, none of these efforts has resulted
`
`in the introduction of a commercially successful high-throughput system capable of producing
`
`production volumes of relatively pure sexed sperm cells having a motility sufficient for effective
`
`20
`
`fertilization.
`
`Accordingly, there is a current need in the animal production industry for a viable high(cid:173)
`
`speed system for efficiently isolating sperm cells based on a specified DNA characteristic (or
`
`other characteristics) to produce quantities of such cells, which can be used on a commercial
`
`scale. Also needed is a sperm handling system that preserves the viability of such isolated
`
`25
`
`sperm as it is processed by the isolating system and that allows for preservation of such isolated
`
`sperm until such time that it is ready for use. The present invention addresses these needs.
`
`This invention also has application to improvements in the field of flow cytometry on a
`
`more general basis. Flow cytometry may broadly be defined as measuring characteristics of
`
`individual particles as they pass generally single file in a fluid stream through a measuring device
`
`30
`
`which, typically, provides information for classifying the particles according to selected
`
`characteristics. Optionally, the particles may then be separated into populations using any
`
`number of techniques, including droplet sorting, droplet interference sorting, and fluid switching.
`
`Another option is to selectively destroy unwanted particles, for example by photo ablation.
`
`In an optically-based flow cytometry system, optics are used to direct and focus a beam
`
`35
`
`of light (e.g., visible light or UV light) on the stream containing the particles, and to collect
`
`emissions from the particles, including scattered light and/or fluorescence emissions from the
`
`particles. In one common optic system, for example, a beam of light (e.g., a laser beam) is
`
`focused on the stream and emissions are collected by a pair of collection units, one positioned
`
`forward of the laser for collecting scattered light emissions and another positioned orthoganally to
`
`40
`
`both stream and the laser for collecting fluorescence emissions. Each collection unit includes a
`
`separate photodetector, which increases the cost of the system. Further, in traditional optic
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`systems the photodetectors translate the collected emissions into electrical signals, which are
`
`analyzed using analog systems to classify the particles according to selected characteristics of
`
`the particles. Analog systems are relatively inexpensive, but only limited information can be
`
`derived from the signals.
`
`5
`
`Others have tried to develop technology that can be used to process sperm cells to
`
`obtain populations of sperm cells that are enriched with sperm that have a desired sex
`
`chromosome. However, the existing technology falls short of the inventive technologies
`
`described herein.
`
`For example, Johnson et al. (U.S. Patent No.5, 135,759) describe the separation of
`
`10
`
`intact X and Y chromosome-bearing sperm populations according to DNA content using a flow
`
`cytometer/cell sorter into X and Y chromosome-bearing sperm enriched populations. As
`
`described, the sperm is combined with a DNA selective dye at a temperature of 30 to 39°C for a
`
`period of 1 hour (39°C} to 1.5 hours {30°C}. A flow cytometer is then used to measure the
`
`amount of fluorescent light emitted as the sperm passes through a laser beam that excites the
`
`15
`
`dye. Because the X chromosome-bearing sperm contains more DNA than the Y chromosome(cid:173)
`
`bearing sperm, with most species of mammal having about 3 to 5% difference, the X
`
`chromosome-bearing sperm emits more fluorescent light than theY chromosome-bearing sperm.
`
`In order to account for the fact that the fluorescence measurement may vary depending on the
`
`rotational orientation of the sperm cells, two photo detectors are used. The first determines
`
`20
`
`whether the sperm cells are properly oriented, while the second takes a measurement that is
`
`used to classify the sperm as having an X or Y chromosome. An oscillator is used to cause the
`
`stream containing the sperm to break into droplets downstream of the place where the sperm
`
`pass through the laser beam. Droplets containing single sperm of a predetermined fluorescent
`
`intensity are given a charge and electrostatically deflected into collection vessels. The collected,
`
`25
`
`gender enriched sperm population, is then used for microinjection, in vitro fertilization, or artificial
`
`insemination.
`Seidel et al. 0NO 02/43574) also describe separation of sperm into gender enriched
`populations of X and Y chromosome-bearing cells using flow cytometry. Seidel et al. describe
`
`staining the cells at a temperature between 30°C and 40°C.
`
`30
`
`United States Patent Application Pub. No. 2003/0157475 A1 {Schenk, August 21, 2003)
`
`describes a method of cryopreserving sperm cells that have been sorted according to X or Y
`
`chromosome content. As noted therein, it is desirable to add a cryoprotectant to sperm cells
`
`before they are cryopreserved to protect the sperm cells during the 'cryopreservation process.
`
`For example, glycerol is one cryoprotectant that is commonly added to bovine sperm cells prior
`
`35
`
`to cryopreservation. However, in order to obtain better protection from the cryoprotectant, it is
`
`desirable to wait for the cryoprotectant to equilibrate with the sperm cells before subjecting the
`
`sperm cells to temperatures below 0°C. During the equilibration period, the cryoprotectant
`
`penetrates the cell membrane to provide intra-cellular protection in addition to any extra-cellular
`
`protection provided by the cryoprotectant. Thus, the cryopreservation methods described in
`
`40
`
`United States Patent Application Pub. No. 2003/0157475 A1 specify that an extender containing
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`3
`glycerol is added to the sperm cells after they have been cooled to about soc. Then the sperm
`cells and glycerol are allowed to equilibrate at soc for anywhere between 1 and 18 hours before
`the sperm cells are subjected to lower temperatures. The disclosure recommends an
`
`5
`
`10
`
`15
`
`equilibration period of between three and six hours in order to obtain the best results.
`
`Unfortunately, the time and expense involved in a 3 to 6 hour equilibration period will
`
`have a negative impact on profitability of a commercial sperm sorting process. Furthermore, in
`
`the context of a commercial sperm sorting process, it is believed that the health of the sperm is
`
`generally improved by reducing the time between collection of the sperm and cryopreservation
`
`(other factors being equal). From this standpoint as well, it would be desirable to have access to
`
`cryopreservation technology that does not require a long equilibration period to obtain the optimal
`
`benefits of a cryoprotectant. Moreover, the known cryopreservation technology i§> reported to
`
`have a detrimental impact on sperm motility, which is indicative of decreased sperm fertility.
`
`Thus, there is a need for cryopreservation techniques that preserves sperm health compared to
`
`conventional techniques.
`
`Summary of the Invention
`
`This invention is directed to an improved system (methods and apparatus) for analyzing,
`
`classifying·and sorting particles based on one or more desired characteristics; the provision of
`
`such a system which, in one embodiment, uses flow cytometry to accurately isolate and sort cells
`
`20
`
`· by DNA content; the provision of such a system which, in certain embodiments, incorporates
`
`sorting protocols which enable the output of the system to be controlled as a function of one or
`
`more factors, including the purity of the desired sorted population of particles, the rate at which
`
`the desired particle population is collected, the loss of desired particles not sorted into the
`
`desired population, and other factors; the provision of such a system which, in one embodiment,
`
`25
`
`operates at high-speed to provide sex sorted sperm for commercial use by the animal production
`
`industry; the provision of such a system which can be used to sort cells without significant
`
`detrimental effect on the cells, including the motility of sperm cells; the provision of a system that
`
`can be used to preserve sorted sperm cells until they are needed with minimal detrimental effect
`
`on the cells, including, the motility of the cells, the provision of such a system which, as it relates
`
`30
`
`to the production of sexed sperm, incorporates techniques which increase the speed and
`
`accuracy of the classification and sorting of the sperm cells; the provision of a flow cytometry
`
`system which uses epi-illumination optics to detect various characteristics of particles to be
`
`analyzed and, optionally, sorted; the provision of such an epi-illumination flow cytometry system
`
`which is economical to manufacture; the provision of a system which, in one embodiment,
`
`35
`
`incorporates multiple flow cytometry units which share an integrated platform for classifying and
`
`(optionally) sorting particles, such as cells in general and sperm cells in particular, at high rates
`
`of production; the provision of such a multi-channel system which share common components
`
`and systems to reduce variations between the channels for more efficient operation; and the
`
`provision of such a sorting system which, in one embodiment, incorporates protocols which
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`enable a sample to be quickly tested to determine the quality of the sample so that the
`
`profitability of further sorting can be evaluated.
`
`In addition, this invention is directed to an improved system (methods and apparatus) for
`
`digitally processing signals representing fluorescence; the provision for such a digital system, in
`
`one embodiment, for detecting analog to digital converted-pulses as a function of background
`
`characteristics; the provision for such a digital system, in one embodiment, for initializing
`
`discrimination parameters; the provision for such a digital system, in one embodiment, for
`
`detecting digital information corresponding to waveform pulses; the provision for such a digital
`
`system, in one embodiment, for digital information analysis including feature extraction; the
`
`provision for such a digital system, in one embodiment, for classifying pulses and defining
`
`5
`
`10
`
`decisions boundaries; the provision for such a digital system, in one embodiment, employing a
`
`droplet break-off sensor to control transducer amplitude; and the provision for using such a digital
`
`system, in one embodiment, to distribute and collect cells for commercial distribution.
`
`Further, this invention is directed an improved comprehensive system (apparatus and
`
`15 methods) for commercial processing of animal semen from the time a semen sample is collected
`
`from a male animal through cryopreservation of a sperm sample containing a greater percentage
`
`of a sperm having a desired chromosome characteristic than exists in the collected semen; the
`
`provision of such a system, in one embodiment, that allows efficient processing of commercial
`
`quantities of viable gender enriched sperm; the provision of such a system that allows, in one
`
`20
`
`embodiment, adjustment of the system to counter day-to-day and animal-to-animal variations in
`
`the semen characteristics; the provision of such a system that, in one embodiment, allows
`
`production of about 18,000,000 gender enriched sperm per hour by a single flow cytometry unit
`
`at 85% purity; and the provision of such a system that allows, in one embodiment, complete
`
`processing of a batch of semen (e.g., the amount of semen collected from a male animal) to yield
`
`25
`
`viable sperm samples having a desired gender characteristic at 85% purity with less than 1 0%
`loss of collected sperm having the desired gender characteristic in about 1 hour of processing
`
`time.
`
`In general, this invention is directed to the apparatus and methods set forth in the claims
`
`of this application.
`
`30
`
`Other objects and features of this invention will be in part apparent and in part pointed
`
`out hereinafter.
`
`Brief Description of the Drawings
`
`Fig. 1 is a work flow diagram for an exemplary sperm sorting process of the present
`
`35
`
`invention;
`Fig. 2 is a schematic diagram of a one embodiment of a flow cytometry droplet sorting
`
`system of the present invention;
`
`Fig. 3 is a side view of a portion of one embodiment of a flow cytometry apparatus of the
`
`present invention for droplet sorting showing an epi-illumination optic assembly focusing an
`
`40
`
`excitation beam on an upward moving fluid stream generated by a nozzle system;
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`Fig. 4 is an end view of one embodiment of a nozzle and nozzle holder of the present
`
`invention;
`
`Fig. 5 is a sectional view of the nozzle and nozzle holder of Fig. 4 taken through cutting
`
`plane 5--5 on Fig. 4;
`
`5
`
`Fig. 6 is a schematic diagram of a sperm cell entrained in a fluid stream being
`
`interrogated by an elliptically shaped beam spot according to one embodiment of the present
`
`invention;
`
`Fig. 7 is a schematic diagram showing the angular envelope for the desired orientation of
`
`a sperm cell in which the light beam from the optics system will strike a wide face of the cell
`
`10
`
`generally broadside;
`
`Fig. 8 is a cross sectional view of one embodiment of a nozzle body of the present
`
`invention;
`
`Fig. 9 is a side view of the nozzle body shown in Fig. 8 showing a series of cutting
`
`planes (A-A through H-H and J-J through K-K) through the nozzle body;
`
`15
`
`Figs. 9A-9H and 9J-9K are sectional views of the nozzle body shown in Figs. 8 and 9
`
`along the corresponding planes (A-A through H-H and J-J through K-K) of Fig. 9;
`
`Fig. 1 0 is a perspective view of a cross section of one embodiment of a nozzle system
`having an orienting baffle in the nozzle;
`
`Fig. 11 is a cross sectional view of the nozzle system shown in Fig. 1 0;
`
`20
`
`Fig. 12 is an enlarged partial cross sectional view of a portion of the nozzle system
`
`shown in Figs. 1 0 and 11;
`
`Fig. 13 is an enlarged partial cross sectional view similar to the view shown in Fig. 12,
`
`but taken from a direction that is perpendicular to the viewing direction in Fig. 12;
`
`25
`
`Fig. 14 is a side view of one embodiment of baffle holder holding a baffle plate;
`Fig. 15 is a top view of the baffle holder and baffle plate shown in Fig. 14;
`Fig. 16 is a top view of one embodiment of a baffle holder rotationally oriented in a
`
`nozzle so that the legs of the baffle plate intersect in a line that is parallel to the major axis of
`
`ellipse D in the nozzle;
`
`Fig. 17 is a top view of one embodiment of a baffle holder rotationally oriented in a
`
`30
`
`nozzle so that the legs of the baffle plate intersect in a line that is perpendicular to the major axis
`
`of the ellipse D in the nozzle;
`
`'
`
`Fig. 18 is a side cross sectional view of one embodiment of a nozzle system including a
`
`baffle showing a series of cutting planes (A-A through E-E) through the nozzle and baffle;
`
`Figs. 18A-18E show the cross sectional flow areas at various points in the nozzle system
`
`35
`
`shown in Fig. 18;
`
`Fig. 19 is a cross sectional view similar to Fig. 12 taken through a nozzle having a baffle
`
`plate that is perpendicular to the longitudinal axis of the nozzle;
`
`Fig. 20 is a cross sectional view of the nozzle shown in Fig. 19 taken through the cutting
`
`plane 20-20 shown on Fig. 19;
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`Fig. 21 is a cross sectional view similar to the cross sectional view of Fig. 18 showing a
`
`nozzle system having a sample introduction conduit at an offset location;
`
`Fig. 22 is a perspective view of one embodiment of a nozzle system mounted on a
`
`nozzle mount of the present invention;
`
`5
`
`Fig. 23 is schematic diagram of a plurality of aligned sperm cells being rotationally
`
`oriented as they pass through an orifice member of the present invention toward the interrogation
`
`location;
`
`10
`
`15
`
`20
`
`25
`
`Fig. 24 is a schematic diagram showing the droplet break-off location downstream from
`
`the nozzle according to one embodiment of the present invention;
`Fig. 25 is a schematic diagram of one embodiment of a break-off sensor system of the
`present invention;
`
`Fig. 26 is a front elevation of one flow cytometry system of the present invention;
`
`Fig. 27 is an enlarged perspective view of a portion of the system shown in Fig. 26 with
`
`parts of the system removed for clarity;
`
`Fig. 28 is a schematic diagram of one embodiment of an epi-illumination optics system of
`
`the present invention;
`
`Fig. 29 is a perspective view of one embodiment of an epi-illumination optics system of
`
`the present invention;
`
`Fig. 30 is a side view of the epi-illumination optics system shown in Fig. 29;
`
`Fig. 31 is a top view of the epi-illumination optics system shown in Figs. 29 and 30;
`
`Fig. 32 is a sectional view of the epi-illumination optics system along the plane 32-32 of
`
`Fig. 30;
`
`Fig. 33 is a sectional view of a portion of the epi-illumination optics system along the
`
`plane 23-33 of Fig. 31;
`
`Fig. 34 is a perspective view showing only elements of the optical filtering system that
`
`are rearward of the dichroic filter of the epi-illumination optics system shown in Fig. 29;
`
`Fig. 35 is a perspective view of another epi-illumination optics system of the present
`
`invention including translational adjustment of the cylindrical lens;
`
`Fig. 36 is a schematic diagram of an interrogation location of one embodiment of the
`
`30
`
`present invention showing a laser beam focused on a fluid stream downstream of the nozzle at a
`
`skewed angle of incidence;
`
`Fig. 37 is a schematic diagram of one embodiment of a sort calibration system of the
`
`present invention;
`
`Fig. 38 is a schematic diagram of one embodiment of an epi-illumination sensor for use
`
`35
`
`with the sort calibration shown in Fig. 37;
`
`Fig. 39 is a block diagram of one embodiment of a digital cell analyzer (DCA) and
`
`processor controller according to the invention.
`
`Fig. 40 is a schematic diagram of one embodiment of a multi-channel sorter of the
`
`present invention showing two channels;
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`Fig. 41 is a work flow diagram of one embodiment of a multi-channel sorter of the
`
`present invention showing four channels;
`
`Fig. 42 is block diagram of one embodiment of an analog cell analyzer (ACA) according
`
`to the invention;
`
`5
`
`Fig. 43 is a graph illustrating a stream of waveform pulses from a photodetector output
`
`detecting fluorescent pulses from cells streaming at an average rate of 10,000 cells/second;
`
`Fig. 44 is an exploded view of Fig. 43 illustrating the stream from a photodetector output
`
`detecting three fluorescent pulses from three cells streaming at an average rate of 10,000
`
`cells/second; a square wave of a 1 OOMHz droplet clock has been superimposed on the
`
`10
`
`illustration to show the synchronization between the three pulses and the square wave pulses of
`
`the droplet clock;
`
`Figs. 45 illustrates movement of a sperm cell relative to a laser beam spot having a
`
`narrow width;
`
`Fig. 49 is an exemplary illustration of the digital information corresponding to a time-
`
`15
`
`varying analog output from a photodetector detecting a single fluorescence pulse based on 122
`
`20
`
`25
`
`samples at a 1 05MHz continuous sampling rate;
`
`Fig. 50 is a schematic diagram illustrating the timing relationship between laser pulses,
`
`fluorescence emissions from a cell resulting from the laser pulses and the digital samples of the
`
`photodetector output in one embodiment of the invention;
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`Fig. 51 is a schematic diagram illustrating how the digital samples shown in Fig. 50 form
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`a pulse waveform;
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`Fig. 52 is a schematic diagram of a pulse waveform from and X sperm cell synchronized
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`with the pulse waveform of a Y sperm cell showing higher peak intensity in the pulse waveform
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`for the X sperm cell;
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`Fig. 53 is a schematic diagram of a pulse waveform showing a threshold and integration
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`window that can be used for pulse analysis;
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`Fig. 54 is a histogram of a sample containing X and Y sperm cells showing the high
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`resolution attainable with slit scanning techniques;
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`Fig. 55 is histogram of a sample containing X and Y sperm cells showing the relatively
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`30
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`poor resolution attained with standard illumination;
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`Figs. 56-59 show fluorescence histograms and scatter plots of peak vs. area for sperm
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`nuclei and live sperm cells;
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`Figs. 60-61 illustrate a four-component model of a fluorescence intensity histogram for
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`sperm cells- Fig. 60 shows raw data and Fig. 61 shows model curves generated by one
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`35
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`embodiment of an iterative algorithm of the present invention based on the data shown in Fig. 60;
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`Figs. 62-63 illustrate a three-component model of a fluorescence intensity histogram for
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`sperm cells- Fig. 62 shows raw data and Fig. 63 shows model curves generated by one
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`embodiment of an iterative algorithm of the present invention based on the data shown in Fig. 62;
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`Fig. 64 illustrates the non-linear nature of the CSD feature; the top panel shows average
`40 M plots for X-bearing andY-bearing sperm cells; the middle panel shows a graph of the first
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`derivatives of these average M plots (i.e. M') for signal amplitude values less than the peak
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`height of the average Y-bearing fluorescence emission pulse; and the bottom panel shows the
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`difference between the first derivatives (M'x- M'v) as a function of signal amplitude;
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`Fig. 65 illustrates one embodiment in which the CSD feature is the computed slope of a
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`5
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`line that passes through two points on the fluorescence emission pulse;
`
`Figs. 66-69 illustrate improved discrimination achieved by use of CSD feature extraction;
`
`Fig. 70 illustrates a bi-variate sort region set on a scatter plot of CSD vs. pulse area
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`10
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`15
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`20
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`25
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`30
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`35
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`scatter;
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`Fig. 71 illustrates one embodiment of flow cytometry re-analyses for a test in which the
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`left panel corresponds to the high recovery/coincident accept sort strategy (no coincidence abort
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`strategy) and the right panel corresponds to the high purity/coincident reject sort strategy
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`(coincident abort strategy);
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`Fig. 72 is a work flow diagram of one embodiment of digital signal processing of the
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`present invention;
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`Fig. 73 is an example of a k-Means clustering strategy that may be employed according
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`to one embodiment of the present invention;
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`Fig. 74 is a conceptual illustration and graphical representation of application of a Bayes
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`Minimum Error decision rule to pulse feature data as may be employed according to one
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`embodiment of the present invention;
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`Fig. 75 is graphical representation of results obtained using a Bayes Minimum Error
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`decision rule and Mahalonobis distance thresh holding as may be employed according to one
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`embodiment of the present invention;
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`Fig. 76 is a conceptual illustration of moving window statistics to provide "forgetting" as
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`may be employed according to one embodiment of the present invention;
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`Fig. 77 is a graphical representation drift compensation as may be employed according
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`to one embodiment of the present invention;
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`Fig. 78 illustrates a fluid stream containing an exemplary distribution of particles;
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`Fig. 79 is a graph showing purity as a function of fluid delivery rate with a coincident
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`accept sort strategy;
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`Fig. 80 is a graph showing the percentage of desired particles successfully sorted into
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`the usable population as a function of fluid delivery rate with a coincident reject sort strategy;
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`Fig. 81 is a graph showing the inverse relationship between the percentage of coincident
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`droplets accepted for sorting into a population of desired particles compared to the percentage of
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`coincident droplets rejected for sorting into that population;
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`Fig. 82 is a decision flow diagram showing the overall operation of one embodiment of a
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`sorting apparatus of the present invention;
`
`Fig. 83 is a side elevation of a cytometer oriented to produce a stream of droplets having
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`a horizontal velocity component and a collection system to collect the droplets;
`
`Fig. 84 is an enlarged perspective view of the collection system shown in Fig. 83 shown
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`40
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`relative to the nozzle system and deflector plates;
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`Fig. 85 is a schematic diagram of one embodiment of a collection system of the present
`
`invention;
`Fig. 86 is a front elevation of an intercepting device of the collection system shown in
`
`Fig. 83;
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`5
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`83;
`
`Fig. 87 is a side elevation of an intercepting device of the collection system shown in Fig.
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`10
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`15
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`Figs. 88-95 show graphical results of several sperm centrifugation experiments;
`Fig. 96-98 are schematic diagrams illustrating the steps in one embodiment of a filtration
`
`method of the present invention;
`
`Fig. 99 is a schematic diagram of one embodiment of a filtration system used to filter
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`sperm cells;
`
`Fig. 100 is a schematic diagram of another filtration system used to filter sperm cells;
`Figs. 101 and 102 show graphical results of sperm cell filtration experiments;
`Fig. 103 is a work flow diagram for one embodiment of a cryopreservation method of the
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`present invention;
`Fig. 104 shows graphical results for a sperm cell cryopreservation experiment;
`Fig. 1 05 is a work flow diagram for one embodiment of a method of processing sperm
`
`cells according to the present invention;
`
`Fig. 1 06 is a perspective view of one embodiment of a multi-channel particle sorter of the
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`20
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`present invention with parts broken away to show internal features of the sorter;
`
`Fig. 107 is a perspective view of a manifold system that may be used for fluid delivery in
`the multi-channel particle sorter of Fig. 1 06;
`Fig. 108 is a perspective view of the manifold system of Fig. 107 showing internal fluid
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`connections of the manifold system;
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`25
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`Fig.