(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`( 43) International Publication Date
`18 August 2005 (18.08.2005)
`
`PCT
`
`(10) International Publication Number
`WO 2005/075629 Al
`
`(51) International Patent Classification7:
`G01N 21/41, 21164
`
`C12N 5/06,
`
`(21) International Application Number:
`PCT /NZ2005/000002
`
`(22) International Filing Date: 19 January 2005 (19.01.2005)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`530972
`
`5 February 2004 (05.02.2004) NZ
`
`(71) Applicant (for all designated States except US): EMBRI(cid:173)
`ONICS LIMITED [NZ/NZ]; Waikato Innovation Park,
`Ruakura Road, Hamilton, 2001 (NZ).
`
`--
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): FRONTIN-ROLLET,
`Andrew [NZ/NZ]; Waikato Innovation Park, Ruakura
`Road, Hamilton 2001 (NZ).
`
`(74) Agents: PIPER, James William eta!.; Floor 1, 29 Water(cid:173)
`loo Road, Lower Hutt, 6009 (NZ).
`
`(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.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`FR, GB, GR, HU, IE, IS, IT, LT, 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).
`
`Declarations under Rule 4.17:
`as to applicant's entitlement to apply for and be granted
`a patent (Rule 4.17(ii)) for the following designations 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,
`
`-iiiiiiii
`[Continued on next page}
`:::: ----------------------------------------------------------------------------------------------
`(54) Title: A METHOD AND APPARATUS FOR ORIENTING SPHERICAL CELLS
`
`!!!!!!!!
`iiiiiiii
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`!!!!!!!!
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`!!!!!!!!
`iiiiiiii
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`iiiiiiii ----
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`22
`
`0\ (57) Abstract: The invention relates to a method that uses the orientation of a sperm cell to determine cell differences due to size,
`~ mass, or density. Cell differences such as differences in DNA mass are used to distinguish X chromosome-bearing sperm cells from
`lf) Y chromosome-bearing sperm cells and therefore have use in in-vitro and in-vivo fertilisation procedures. The orientation of indi(cid:173)
`t"-- vidual sperm cells is determined by measuring non-fluorescent light. The method uses one detector to measure the magnitude of
`0
`fluorescence (for DNA (sex) measurement from the flat surface of the spermatozoon), and a second detector to measure the magni(cid:173)
`ln tude of refracted non-fluorescent light derived from a separate light source. The separate light source is derived from part of a phase
`0
`contrast or Dark field optical system to provide orientation data. Importantly, all excitation and fluorescent light is excluded from the
`0
`second detection system by band-pass optical filters thereby providing for a cleaner signal from the concave edge (no fluorescence
`M signal from the flat surfaces of the spermatozoon). It is reported that the orientation of a sperm cell when determined by passing
`0 light using optical phase contrast or Dark field optics through a sperm cell of interest provides for improved efficiencies, improved
`> processing speeds and increased reliability in the results obtained. A further aspect of the invention describes an immobilising or
`
`~ ablative laser for immobising or destroying unwanted sperm.
`
`

`

`WO 2005/075629 Al
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`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, LV, 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, SM, SY,
`TJ, TM, TN, TR, TT, TZ, VA, UG, UZ, VC, VN, YU, ZA,
`ZM, ZW, ARIPO patent (BW, GH, GM, KE, LS, MW, MZ,
`NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian patent (AM,
`AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT,
`BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl, FR, GB, GR,
`HU, IE, IS, IT, LT, LV, MC, NL, PL, PT, RO, SE, Sf, SK,
`TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG)
`
`as to the applicant's entitlement to claim the priority of the
`earlier application (Rule 4. I 7( iii)) for all designations
`ofinventorship (Rule 4.17(iv))for US only
`
`Published:
`with international search report
`
`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.
`
`

`

`wo 2005/075629
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`PCT /NZ2005/000002
`
`A METHOD AND APPARATUS FOR ORIENTING ASPHERICAL CELLS
`
`FIELD OF INVENTION
`
`The present invention generally relates to a method and apparatus for orienting desired cells, or parts
`
`of cells, preferably, desired sperm cells and, more particularly, the invention relates to a method and
`
`apparatus for orienting, selecting and retaining viable desired sperm cells.
`
`BACKGROUND OF THE INVENTON
`
`There has been a long felt need for a reliable, qualitative, quantitative and cost-effective method for
`
`selecting sperm, which may be used to produce animals of a desired sex
`
`In particular, in the livestock industry farmers or breeders require cows, pigs, sheep, goats, deer,
`
`buffalo, horses, etc which are of a preferred sex. For example, bulls are of limited use to a dairy
`
`farmer, whereas pig farmers have long been aware that female pigs grow at a faster rate than their
`
`male counterparts.
`
`Similarly, cattle and sheep farmers understand only too well that the males of these species produce
`
`meat at a faster rate than females.
`
`In mammals the egg carries only the X chromosome whereas the sperm carries either an X or a Y
`
`chromosome. The sex of progeny is therefore determined by the sperm cell. When a sperm and an
`
`egg are combined and the sperm carries the X chromosome the offspring is female (XX). However, if
`
`the sperm carries the Y chromosome, once combined with the X chromosome carried by the female
`
`the resultant offspring will be male (XY).
`
`In sperm there is a known difference in DNA content between the X (larger) and theY (smaller) sperm
`
`of for example 3.4% in pigs, 3.9% in cattle and 4.2% in sheep. This measurable difference can be
`
`used to determine the sex of the sperm, that is, if it is an X chromosome (female) or if it is a Y
`
`chromosome (male) bearing sperm.
`
`The prior art discusses and provides for methods for sorting mammalian sperm into X and Y
`
`populations. However, the only reliable methods that maintain sperm viability post-analysis describe
`
`the measurement of the DNA mass of individual sperm. These methods essentially use a modified
`
`flow cytometer utilising fluorescence measurement to detect what are essentially small differences
`
`between the X andY sperm, wherein the sperm pass single file through a system that measures the
`
`DNA content of individual sperm.
`
`Some techniques have been expanded to use a bevelled sample injection tip and a second
`
`fluorescence detector in a forward position. This second fluorescence detector is adapted to
`
`1
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`wo 2005/075629
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`PCT /NZ2005/000002
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`determine the orientation of flat oval shaped sperm heads with respect to the first detector as they
`
`pass through the system.
`
`In both cases it is the magnitude of fluorescence that is being measured. This requires two separate
`
`fluorescence detectors, or at the very least two discrete fluorescence readings.
`
`Further adaptations allow for those unwanted sperm to be gated and pass through as waste and
`
`discarded.
`
`The prior art therefore describes a flow cytometric system, which requires two separate measurements
`
`of the magnitude of fluorescence of the sperm cell, one to determine the sex of the sperm, the other to
`
`determine the sperm's orientation. Those skilled in the art would recognise that due to the morphology
`
`of sperm cells (flat ovoid shape) and extremely high refractive indices, it is not possible to accurately
`
`measure the DNA content of sperm unless said sperm are correctly oriented to the DNA fluorescence
`
`detector.
`
`The prior art methods have proven to be expensive - and do not always provide for routine
`
`efficiencies much in excess of 80%, although 95% efficiencies have been reported. Furthermore,
`
`previously used methods can sometimes overload the photomultiplier tube resulting in a relatively high
`
`background noise to signal ratio and an unacceptably high number of incorrectly sexed sperm.
`
`Johnson and Pinkel teach in Cytometry 7: 268-273 (1986), of the provision of two fluorescence
`
`detectors, one at 90 degrees and a second at 0 degrees. These detectors simultaneously collect
`
`fluorescence signals from the edge and flat side of the sperm nucleus. The fluorescent detector at 90
`
`degrees is used to determine the orientation of an individual spermatozoon orthogonal to a second
`
`fluorescence detector, which measures the magnitude of fluorescence and hence total DNA content
`
`(and thereby sex) of the spermatozoon.
`
`The prior art disclosed by Lawrence Johnson in US 5, 135,759, and assigned to XY Inc., teaches of a
`
`method, which measures the magnitude of fluorescence from both detectors to provide relevant data.
`
`That is, fluorescence is used to determine both orientation and the DNA content (sex) of any given
`
`sperm cell. This Johnson Patent does not visit the novel concept of determining orientation using
`
`refracted non-fluorescent light emission.
`
`The Johnson method/apparatus is based solely around a modified flow cytometer. The flow cytometer
`
`is a commonly used laboratory instrument for the analysis of individual cells and separates the cells
`
`into three populations. Essentially the flow cytometer injects cells into a sheath fluid system that
`
`teases cells out into single file and orients them within an optical/focal plane. Dependent upon internal
`
`2
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`PCT /NZ2005/000002
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`geometry, the nozzle may also orient cells radially within the sheath fluid flow.
`
`The cells are then ejected under pressure from the nozzle in a stream of droplets, each droplet ideally
`
`containing a single spermatozoon (some droplets contain multiple spermatozoa and some none).
`
`Individual spermatozoa are typically optically analysed within the droplets and by means of applying a
`
`positive, negative or zero charge to individual droplets, according to analysis, and then passing said
`
`droplets between electrically charged deflection plates, sorting into separate populations may be
`
`accomplished. Without going into detail the process can be problematic, particularly at high speed.
`
`Nevertheless XY Inc. claim sort purities of 90-98% dependent on processing speeds, i.e. the higher
`
`the speed the lower the accuracy of sorting.
`
`A significant disadvantage of the Johnson/XY, Inc. process is the viability of the sorted spermatozoa.
`
`Droplets exit the nozzle at high speed and dependent upon sorting speeds, droplet velocity may reach
`
`speeds of 20 metres per second resulting in huge stresses upon the spermatozoa impacting upon
`
`fluids in, or the walls of, the collection vessel. Streaming of tiny droplets into air also exposes
`
`spermatozoa to oxidative stress and it is thought that such stresses may affect sperm viability and
`
`result in relatively lower yields of viable sperm.
`
`Various nozzle systems are disclosed in US 6,263,745, US 6,357,307 & US 6,604,435. These
`
`documents form differing aspects of the same invention. They all relate to an improved nozzle system
`
`for a flow cytometer and generally describe a means to accelerate the delivery of sperms cells,
`
`hopefully at the correct orientation, to be sorted and analysed. US 5,985,216. This document
`
`describes a tapered sorting nozzle. It is reported to be able to both orient and allow for sorting of
`
`desired and viable sperms types from a sample. None of the above documents disclose the novel
`
`aspects of the present invention.
`
`WO 98/34094 teaches of an epillumination system adapted to a flow cytometer that does not require
`
`sperms to be aligned or oriented. In effect it organises and directs the collection of fluorescent light
`
`from an illuminated sample stream in a flow cytometer by using a paraboloid or ellipsoid shaped
`
`collector. The '094 method gives comparatively slow passage flows and may compromise cell
`
`viability.
`
`WO 01/85913 describes a method of analysing the DNA volume of X andY carrying sperm. The
`
`document discusses the use of electromagnetic radiation (or simply light which is electromagnetic
`
`radiation) and modified differential interference contrast optics to measure a sex differentiation
`
`characteristic such as volume of sperm cell heads. The electromagnetic radiation can be a laser,
`
`microwave or UV light. The thrust of the '913 document attacks the problem of orientation, distorted
`
`readings and background signals caused by fluorescence measurement. The document states that
`
`this "can allow small differences in photoemissive light to be differentiated even when total light
`
`emitted from each photoemissive event is high, or even when there are a high number of bright serial
`
`3
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`events per second". The '913 patent measures minute changes in phase shift, ie the difference
`
`between the waveform characteristics of light prior to and after penetration of the sperm. The
`
`document teaches of the use of complicated, modified interference optics and polarised light to
`
`determine sample orientation. The use of phase contrast or Dark field optics to measure refracted
`
`non-fluorescent light to determine sample orientation is not contemplated.
`
`There is a need therefore for a simple and effective method and apparatus, which enables individual
`
`cells to be sorted accurately and quickly from a population of cells and wherein the cells remain viable.
`
`OBJECT OF THE INVENTION
`
`It is an object of the present invention to provide an improved method and/or apparatus for selecting
`
`desired cells, or parts of cells, or is one which will obviate or minimise the foregoing disadvantages or
`
`will at least provide the public with a useful choice.
`
`STATEMENT OF THE INVENTION
`
`Accordingly, a first aspect of the invention provides for a method of determining the orientation of a
`
`cell in a process wherein said orientation is used to allow for the determination of cell differences due
`
`to size, mass, volume or density and whereby the orientation of the cell is determined by measuring
`
`non-fluorescent light.
`
`Preferably, the orientation is determined by measuring light using a band pass filter to exclude all light
`
`other than from a phase contrast optical system or a system utilising Dark field optical techniques.
`
`Preferably, the cell is an aspherical cell.
`
`Preferably, the cell is a sperm cell.
`
`Preferably, the method for determining the orientation of the cell does not require the cell to be
`
`encapsulated within a droplet
`
`Preferably, the method for determining the orientation of the cell is used in tandem with a method for
`
`measuring the DNA content of the cell.
`
`Preferably, the method for determining the orientation of the cell is used simultaneously with a method
`
`for measuring the DNA content of the cell.
`
`Preferably, the method for determining the orientation of the cell is used in a method for selecting
`
`sperm of a desired chromosome complement.
`
`Preferably, the method for determining the orientation of the cell is further used in a method for
`
`4
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`PCT /NZ2005/000002
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`differentiating X chromosome bearing sperm from Y chromosome bearing sperm and/or selecting a
`
`population of cells having a desired sex.
`
`Accordingly, a second aspect of the invention provides for a method of selecting a desired cell, or
`
`parts of a cell, the method having the following steps:
`
`(i)
`
`(ii)
`
`(iii)
`
`(iv)
`
`(v)
`
`(vi)
`
`(vii)
`
`passing suitably maintained cells from a sample of cells of interest into a testing zone,
`
`exposing said cell sample of interest to a first light source having a first wavelength,
`
`exposing said cell sample of interest to a second different light source of a second different
`
`wavelength,
`
`collecting light energy emitted at (ii) and (iii) above,
`
`analysing the light collected at (iv) to determine whether the desired predetermined
`
`parameters are met,
`selecting those cells, or parts of cells, which meet said desired parameters,
`
`collecting the selected cells in a suitable viability maintenance medium, and/or
`
`(viii)
`
`eliminating those unwanted cells, or parts of cells, as waste.
`
`Preferably, the cells are sperm cells.
`
`Preferably, the sperm cells are stained with a suitable DNA-specific binding fluorochrome.
`
`Preferably, the first light source is of a suitable wavelength(s) to excite fluorescence in said DNA
`
`specific binding fluorochrome(s).
`
`Preferably, the first light source develops one or more wavelengths of emitted fluorescent light to
`
`enable analysis of the DNA content of a sperm cell.
`
`Preferably, the fluorochrome is selected from SYBR green I, SYBR green II, SYBR gold, and
`
`Bisbenzimide H33342
`
`Preferably, the second light source is used to determine the orientation of the cell.
`
`Preferably, the second different light source uses a light source derived from a phase contrast optical
`
`system or one using Dark field optical techniques.
`
`Preferably, the cell is simultaneously exposed to said first light energy and second different light
`
`energy.
`
`Preferably, the cell is passed through an orientation device wherein the orientation of the cell is
`
`hydrodynamically oriented to achieve a uniform radial geometry with respect to the detector(s)
`
`Preferably, the testing zone is a rectangular receiving area adapted to maintain the orientation of
`
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`single cells, most preferably sperm cells during analysis.
`
`Preferably, the cells to be tested are delivered to the rectangular testing zone at a flow rate, sufficient
`
`to maintain and retain cell viability, preferably at above 1,000 cells per second, and most preferably
`
`between 1,000 to 100,000 cells per second.
`
`Preferably, the cells to be tested are delivered to the rectangular testing zone at a flow rate of from
`
`5,000 to 40,000 cells per second.
`
`A third aspect of the invention provides for an apparatus for selecting a desired cell, or parts of a cell,
`
`the apparatus comprising:
`
`(i)
`
`a means for passing suitably maintained cells from a sample of cells of interest into a testing
`
`zone,
`
`(ii)
`
`a means of exposing said cell sample of interest to a first light source having a first
`
`wavelength,
`
`(iii)
`
`a means of exposing said cell sample of interest to a second different light source having a
`
`different wavelength,
`
`(iv)
`
`separate means for collecting and, if necessary, amplifying light emitted by said sample at (ii)
`
`and (iii)
`
`(v)
`
`a means for analysing the data collected by separate means (iv) to determine whether desired
`
`predetermined parameters are met,
`
`(vi)
`
`a means for selecting, collecting and maintaining cells in viable condition meeting said desired
`
`predetermined parameters, and/or
`
`(vii)
`
`a means for eliminating, those unwanted cells, or parts of cells, as waste.
`
`Preferably, said first light source is a source of electromagnetic radiation, such as a laser.
`
`Preferably, said first light source is adapted to allow for the analysis of the DNA content of a cell.
`
`Preferably, said second light source is derived from a phase contrast optical system or a system
`
`utilising Dark field optical techniques.
`
`Preferably, said second light source is adapted to determine the orientation of a cell.
`
`Preferably, said means for collecting light emitted from said sample after exposure to said first light
`
`source comprises one or more microscope objective(s), or similar.
`
`Preferably, said means for collecting light emitted by said sample after exposure to said second light
`
`source is an optical detection system adapted to collect light energy of a non-fluorescent wavelength.
`
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`Preferably, said analysis and identification means is a multi-channel analyser or computer
`
`programmed with suitably developed computer software.
`
`A fourth aspect of the invention provides for a delivery device for delivering in a laminar flow suitably
`
`maintained sperm cells from a sample injection tube via a hydrodynamic radially orienting nozzle and
`
`thereafter to a testing zone, the delivery device comprising:
`
`an elongated tube having a first end portion and a second end portion,
`
`the first end portion comprising a nozzle,
`
`the second end portion comprising a pre-collection or deceleration zone, and wherein,
`
`the first and second end portions are spaced apart either side of a substantially rectangular cross(cid:173)
`
`sectioned testing zone and wherein,
`
`said first end portion comprising said nozzle has a first end and a second end, said first end adapted
`
`to communicate with a sample injection tube to receive said sample and said second end being
`
`contiguous with said testing zone, the nozzle being of a size and shape sufficient to maintain said
`
`sperm cells in·a laminar flow at a hydrodynamic radial orientation, and
`
`said second end portion comprising said pre-collection or deceleration zone is configured to convey
`
`sperm cells to a collection means such that said cells after exiting the testing zone are maintained in a
`
`viable condition suitable for use in an in-vitro or in-vivo fertilisation procedure.
`
`Preferably, the pre-collection or deceleration zone is flared outwards from the substantially rectangular
`
`cross-sectioned testing zone.
`
`Preferably, in use, as the cells pass from the injection tube and into the delivery device the orientation
`
`nozzle orients and maintains individual cells into a position which allows for each individual cell to
`
`pass through a first light source having a first wavelength and light emitted by said cell to be detected
`
`and analysed for DNA mass, and which simultaneously allows for said cell to pass through a second
`
`different light source having a second different wavelength to be detected and analysed for correct
`
`orientation.
`
`Preferably, individual cells are exposed to said first and second light sources simultaneously.
`
`A fifth aspect of the invention further provides for a method of selecting a desired sperm cell, or part of
`
`a sperm cell, the method having the following steps:
`
`(i)
`
`(ii)
`
`staining intact, viable sperm collected from a male mammal with a suitable fluorescent
`
`dye, such that the DNA takes up the fluorescent dye uniformly,
`
`maintaining the stained sperm in a suitable maintenance medium sufficient to maintain the
`
`sperm and/or contained DNA within the cell in a viable condition,
`
`(iii)
`
`passing the maintenance medium containing the sperm before a suitable excitation light
`
`source causing the stained DNA to fluoresce,
`
`-
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`7
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`PCT /NZ2005/000002
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`(iv)
`
`passing the maintenance medium containing the sperm through both a means for
`
`measuring the fluorescence of the stained DNA and a means for detecting the orientation
`
`/
`
`of the sperm,
`
`(v)
`
`collecting light energy emitted by said sperm cell, converting the light energy into electrical
`
`signals and analysing the electrical signals via a multi-channel analyser or suitably
`
`programmed CPU,
`
`(vi)
`
`selecting those sperm cells, or parts of sperm cells meeting desired predetermined
`
`criteria, and
`
`(vii)
`
`a means for eliminating those cells, or parts of cells, which fail to meet the desired
`
`predetermined criteria.
`
`SUMMARY OF THE INVENTION
`
`The prior art disclosed by Lawrence Johnson in US 5, 135,759, and assigned to XY Inc., teaches of a
`
`method, which uses both a 90 degree and 0 degree optical detector to collect and measure the
`
`magnitude of fluorescence to determine both orientation and the DNA content (sex) of any given
`
`sperm cell.
`
`By contrast, the present invention provides for a novel method, which uses a first detector to measure
`
`the magnitude of fluorescence for DNA measurement from the flat surface of the spermatozoon), and
`
`a second detector to measure the magnitude of refracted non-fluorescent light derived from a separate
`
`light source. The separate light source is derived from part of a phase contrast or Dark field optical
`
`system to provide orientation data. Importantly, all excitation and fluorescent light and any unwanted
`
`or aberrant light from any other sources is excluded from the second detection system by appropriate
`
`band-pass optical filters thereby providing for a cleaner signal from the concave edge (ie any
`
`fluorescence signal emitted from the flat surfaces of the spermatozoon is excluded and not
`
`measured). The use of phase contrast or Dark field optics to measure said non-fluorescent light
`
`achieves a significant lesser loading of the PMT. This reduction in PMT loading therefore allows for
`
`higher processing speeds, economies in processing costs and significantly higher sperm viability
`
`retention due to shorter individual sample processing time. The Johnson method is speed limited as
`
`higher processing speeds can result in an undesirable high background noise to signal ratios created
`
`by signal bounce.
`
`Surprisingly, the present inventor has found that a process wherein the orientation of a sperm cell is
`
`determined by passing light using optical phase contrast or Dark field techniques through a sperm cell
`
`of interest provides for improved efficiencies and increased reliability in the results obtained. In other
`
`words orientation of sperm cells - the correct orientation defining whether a result should be accepted
`
`for further analysis - can be determined by measuring non-fluorescent light emitted by a sperm cell.
`
`The use of phase contrast optics or Dark field optical techniques as a means to measure refracted
`
`non-fluorescent light has never before been considered as a means to determine the orientation of
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`wo 2005/075629
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`aspherical cells.
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`PCT /NZ2005/000002
`
`The inventor has also found unlike the prior art that there is no need for the cells of interest to be
`
`encapsulated or confined within an electrically charged medium during the analysis and collection
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`phase of the process. Previously, once the DNA content of a sperm cell had been determined, the
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`cells were encapsulated in a droplet to which is appended an electric charge, the charge being
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`dependent on a cell's X or Y sex chromosome content. The droplets were then separated based
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`upon the charge they received. The present invention simply selects those cells having a desired sex
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`chromosome based upon predetermined parameters programmed in the analyser. If the criteria are
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`met the cell passes untreated and is retained in a viable condition suitable for either in-vivo or in-vitro
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`fertilisation uses. If the criteria are not met, the cell may be permanently immobilised through
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`permeabilisation of the plasma membrane by heat transference generally, but not necessarily, by
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`exposure to a laser or, partially or completely destroyed by a process of ablative photodecomposition,
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`generally by exposure to a laser.
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`This invention also teaches the use of a rectangular testing zone located downstream of an orienting
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`nozzle. A cell emerging from the orienting nozzle can be maintained at the correct radial orientation to
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`allow for accurate DNA analysis. The substantially rectangular configuration of the testing zone of the
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`invention has been found to provide for superior accuracy and more reliability in the results obtained.
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`Previous testing processes maintain the cell being measured in a circular cross sectional fluid stream
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`or liquid droplet, which is of an essentially elliptical or circular configuration as the cell emerges from
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`the nozzle. This configuration, although allowing for commercially acceptable cell flow rates of a
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`desired orientation, also allows for inaccuracies due to light being refracted from the curved surfaces
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`of the fluid stream or droplets being measured.
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`Importantly, this rectangular testing zone provides for four flat surfaces. The improvement results in a
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`significant reduction in unwanted refracted light over systems where curved surfaces are used thus
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`eliminating false readings. As such the provision of four flat surfaces provides for a much-improved
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`reliability over previously disclosed systems.
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`The present invention therefore comprises at least four novel components, the aspects of which will be
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`outlined later in greater depth. Firstly, the invention uses phase contrast or Dark field optics to
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`determine a desired cells orientation with respect to a DNA measurement detector. Secondly, the
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`invention makes no requirement for the cells of interest to be encapsulated in droplets or otherwise to
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`enable desired cells to be physically separated from those that are not wanted. Thirdly, the use of a
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`substantially rectangular testing zone reduces the effects measurement of unwanted light has on the
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`process. Fourthly, the invention teaches of a laser actuated means for temporarily or permanently
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`immobilising or even destroying unwanted cells.
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`The above features therefore provide significant, surprising and novel advantages over existing cell
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`PCT /NZ2005/000002
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`selection/sorting processes and in particular over those processes directed to the sexing of sperm
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`cells.
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`DETAILED DESCRIPTION OF THE INVENTION
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`The following examples are illustrative only and, where specific integers are mentioned which have
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`known equivalents, such equivalents are deemed to be incorporated herein as if individually set forth.
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`The examples describe preferred embodiments only and are non-limiting.
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`The present application has particular relevance in the selection of sperm cells carrying a desired sex
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`chromosome. The ability to provide for populations having viable X chromosome bearing sperm or Y
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`chromosome bearing sperm at purity of 95% or even 98% or more is now achievable.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Various other objects and features and attendant advantages of the invention will become more fully
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`appreciated as the same becomes understood in conjunction with the accompanying drawings, in
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`which like reference characters designate the same or similar parts throughout several views, and
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`wherein
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`Figure 1 is a schematic showing the system methodology.
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`Figure 2 is a flow chart of the general process.
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`Figure 3 illustrates the delivery device.
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`Figure 4 illustrates the injection tube, delivery device and collection point relationship.
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`Figure 5 shows the cross-sectional relationship of components comprising the apparatus as seen
`through line 'A- A' of figure 4.
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`Figure 6 provides an overview of the components comprising the apparatus.
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`Example 1
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`Turning now to figures 1 to 6, the various apparatus used and method steps involved in the process
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`are described in detail.
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`Live sperm to be differentiated according to their sex characteristic are collected by standard collection
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`techniques and maintained in a suitable medium such as a Tris buffer medium. The DNA within the
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`cells is stained with a non-toxic fluorochrome, preferably SYBR green I, SYBR green II, SYBR gold or
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`Bisbenzimide H 33342.
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`Intact stained sperm are then subjected to a fluorescence excitation energy
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`source provided by an optical fibre or hollow glass fibre (26). The preferred excitation wavelength is
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`PCT /NZ2005/000002
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`about 488-497mm and is dependent on the particular fluorochome being used. Signals emitted are
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`collected via a fluorescence collection objective (11) or similar, measured by a photo multiplier tube
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`(PMT) (18A) and processed/analysed by