Patent Description:
Flow cytometry may utilize light detection to assess characteristics of particles, such as cells, flowing through the cytometer. In certain applications, a cytometer may detect light emitted by cells, including light emitted by fluorescent, DNA-intercalating dye. The ability to assess and classify particles is a fundamental operation of cytometers.

Cytometers measure characteristics of particles and provide information on the measured characteristics. Typically, this information is represented graphically in the form of a histogram or a scatter plot, although this information is usually also collected and stored in a computer-readable form. The information relating to these measurements can be used to identify subpopulations within the starting set of particles, or to define subjective or objective values for the measured characteristics. This is often accomplished by defining a gate using the graphical representation. Generally, the procedure for drawing a gate around a population is done manually: an operator makes a visual inspection of the graphical representation of the mixture of particles as it is analyzed, and manually sets the points used to differentiate populations or subsets of the mixture from one another. Once subsets have been defined by the drawing of gates, all subsequent events can be defined as falling within a gate (i.e. as belonging to the defined subset) or as falling outside a gate (i.e. not belonging to the defined subset, and/or belonging to a different subset). This has significance to a number of applications, including quantitative flow cytometric assays, and cellular discrimination such as fluorescence-activated cell sorting (FACS). A method for identifying subpopulations within a mixture of particles is disclosed in <NPL>.

One such application is the determination of whether a sperm cell has an X-chromosome (which will produce a female zygote) or alternatively an Y-chromosome (which will produce a male zygote). A sperm cell with two X-chromosomes may have approximately <NUM>% more DNA than a sperm cell with an X-chromosome and a Y-chromosome. By identifying the chromosomal content of a sperm cell, it may be possible to create a relatively high-purity population of X-chromosome or Y-chromosome bearing sperm cells. Such a population may be generated, for example, by keeping the desired gender and killing the undesired one (or by segregating the two genders). The substantially high-purity population may be used to inseminate a female animal (such as bovids, equids, ovids, goats, swine, dogs, cats, camels, elephants, oxen, buffalo, or the like) to obtain a desired male or female offspring, with relatively high probability.

The characteristics of the chromosomes may be identified by staining the DNA of sperm cells with a fluorochrome (for example, a DNA-intercalating dye). The stained sperm cells may be forced to flow in a narrow stream or band and pass by an excitation or irradiation source such as a laser beam. As the stained sperm cells (a plurality of particles) are irradiated, the fluorochrome in the plurality of particles emits a responsive fluorescent light. The amount of fluorescent light may vary based at least in part on a relative amount of at least one particle differentiation characteristic (for example, the relative amount of DNA indicative of the presence of an X or Y chromosome) present in each of the plurality of particles. The fluorescent light may be received by one or more optical elements that ultimately focus the received light onto a detection component, such as a photomultiplier tube ("PMT") or an avalanche photodiode (APD). The detection component may generate an electrical, analog signal in response to the received light. The analog signal may vary in correspondence with the amount of received light. This signal may then be processed (for example, digitized and analyzed by a processor) to assess the chromosomal content of the sperm cell, which can be represented graphically as a histogram (e.g. data as a vector of peak voltage values, with the index adjustment being a function of bin width) or a scatterplot (e.g. data as a mapping of peak voltage values and area voltage values into a vector that is used to display the most current data as a spectrogram). These graphical representations can then provide the basis for defining subpopulations and/or drawing gates. This is typically done by a user, using her best judgment to pick points establishing the boundary of a gate or a threshold.

Currently available systems for setting gates or thresholds for defining subpopulations suffer from several significant deficiencies. First, the selection of the definition criteria (i.e. the values at which the gate or threshold points are set) is performed by individual operators. This is fundamentally subjective and variable among operators, and even between samples run by the same operator, or between runs of the same sample by the same operator. This ultimately leads to variability in the operation of the flow cytometric systems. In the case of sex sorting cytometers, this can result in significant differences in the yield (i.e. the number of live cells collected) or purity (i.e. the proportion of collected cells having an X-or Y-chromosome) of the sex sperm cell product.

In addition, current approaches to sub-population definition and discrimination result in operation where the bases for discrimination are essentially static-the gates or thresholds remain unchanged during operation. In practice, this means that an operator must monitor the subpopulations over the course of the run, and/or the relationship of the gates or thresholds to a subpopulation may change during the course of operation, due to a number of factors such as changes in temperature or pressure, or loss of signal.

The systems and techniques described herein overcome these and other shortcomings and provide significant benefits, as will be appreciated by those skilled in the art.

According to certain inventive implementations, the present invention provides a system for identification of sub-populations within a mixture of particles according to claim <NUM>. According to certain implementations, the system includes a sample delivery apparatus, a cytometer, a control apparatus, and a sorting apparatus. The control is configured to implement a self-calibrating identification or discrimination technique. The self-calibrating identification or discrimination technique utilizes the information obtained for particles in the mixture to define subpopulations within the mixture. The system then assigns a subpopulation classification to particles in the mixture based on this definition. The definition of subpopulations is continuously refined though the integration of additional information obtained for additional particles in the mixture. The control is further configured to receive event data from the cytometer in the form of values corresponding to detected particles in a sample, distinguish the values of measured characteristics for each sub-population in the sample, assign a critical range of values to each of the sub-populations based on the distinguishing, updating the critical range of values based on the distinguishing of the values of the measured characteristics for sub-populations in a second set of particles, and assign a sub-population classification to particles in the mixture based on the updated critical range of values.

According to certain inventive implementations, the control may further be configured to identify at least one critical value for a first characteristic from the distribution; obtaining the values for a second characteristic associated with the critical value for the first characteristic; produce a distribution of the values for the second characteristic; identify at least one critical value for the second characteristic from the distribution of values for the second characteristic; co-locate the critical values for the first and second characteristics in a feature space; and determining the at least one discrimination values from the critical values co-located in the feature space.

According to certain inventive techniques, a method includes: identifying subpopulations within a mixture of particles, comprising: measuring a characteristic of a first set of particles in a mixture of particles having two or more sub-populations, each sub-population being distinguishable by the characteristic; distinguishing the values of the measured characteristics for each of the sub-populations in the mixture; assigning a critical range of values to each of the sub-populations based on the distinguishing; subsequent to the assigning a critical range of values, measuring the characteristics of a second set of particles in the mixture of particles; subsequent to measuring the characteristics of a second set of particles in the mixture of particles, distinguishing the values of the measured characteristics for each of the sub-populations in the second set of particles; updating the critical range of values based on the distinguishing of the values of the measured characteristics for each of the sub-populations in the second set of particles in real time; and assigning a sub-population classification to particles in the mixture based on the updated critical range of values.

According to certain inventive techniques, the methods further comprise sorting said particles according to the classification assigned to the particles based on the updated critical range of values. According to certain inventive techniques, the methods further comprising destroying or incapacitating the sub-population assigned the classification. According to certain inventive techniques, the particles are sperm cells. According to certain inventive techniques, the sperm cells are bovine sperm cells. According to certain inventive techniques, the characteristic of the sperm cells is the presence of an X-chromosome or a Y chromosome. According to certain inventive techniques, the sperm cells have been stained with a fluorescent DNA-binding dye.

According to certain inventive techniques, a method includes identifying multiple sets of particles within a mixture of sets of particles, each sub-population having a characteristic different from other sub-populations, comprising: providing the mixture of particles; detecting a characteristic of a first set of multiple particles within the mixture to produce a signal indicative of the characteristic for the first set of particles; generating event data for the signal indicative of the characteristic for said first set of particles; compiling the event data for the first set of particles; generating a distribution of event data for the first set of particles; calculating at least one discrimination value that defines a sub-population of particles based on the distribution of event data for the first set of particles, wherein calculating at least one discrimination value comprises defining at least on critical value related to a detected characteristic; adjusting the at least one critical value to include event data from a second set of particles in real time; and classifying particles based on the at least one discrimination value that defines the subpopulation. According to certain inventive techniques, the method further comprises instructing a sorting apparatus to include or exclude events based on the classification. According to certain inventive techniques, the method further comprises destroying or incapacitating the sub-population assigned the classification. According to certain inventive techniques, the particles are sperm cells. According to certain inventive techniques, the sperm cells are bovine sperm cells. According to certain inventive techniques, the characteristic of said sperm cells is the presence of an X-chromosome or a Y-chromosome. According to certain inventive techniques, the sperm cells are labeled with a fluorescent DNA-binding dye.

According to certain inventive techniques, calculating at least one discrimination value further comprises: identifying at least one critical value for a first characteristic from the distribution of event data for the first set of particles; obtaining the values for a second characteristic associated with the critical value for the first characteristic; producing a distribution of the values for the second characteristic; identifying at least one critical value for the second characteristic from the distribution of values for the second characteristic; co-locating the critical values for the first and second characteristics in a feature space; and determining the at least one discrimination values from the critical values co-located in the feature space. According to certain inventive techniques, the critical values comprise one or more peak characteristic values for a first sub-population, and one or more peak characteristic values for a second subpopulation. According to certain inventive techniques, the peak characteristic values comprise peak fluorescence intensity and peak total fluorescence. According to certain inventive techniques, the critical values further comprise a trough value between the peak characteristic values for the first and second subpopulations. According to certain inventive techniques, the discrimination value comprises a gate.

According to certain inventive techniques, a sexed sperm cell sample is produced by any one of the methods of any of the preceding claims. The foregoing summary, as well as the following detailed description of certain techniques and systems of the present application, will be better understood when read in conjunction with the appended drawings. For the purposes of illustration, certain systems and techniques are shown in the drawings. It should be understood, however, that the claims are not limited to the arrangements and instrumentality shown in the attached drawings.

Certain inventive systems and techniques discussed herein relate to a cytometer that includes a mechanism to use known relationships in frequency or quantum of characteristics of particles in a mixture to identify, define, or distinguish subpopulations within the mixture. The advantageous systems and techniques allow for optimization of throughput (yield and/or purity), reduction in variability and increase in operational consistency, and reduction in human user input and variability associated therewith. These advantageous systems and techniques provide decisions that reliably discriminate between particles having different characteristics, thereby providing differential classification.

The systems and methods of the present invention may be used for discrimination of one or more subpopulations of particles in a mixture. This can be accomplished by detecting or assessing a single characteristic, or multiple characteristics of the particles. Characteristics can be assessed using different means of identification, including, for example, fluorescent markers (e.g. fluorescent dyes or fluorescent markers coupled to one or more target-specific molecules, such as antibodies or antibody fragments or other molecular markers). In a further aspect, the methods and systems of the present invention involve sorting cells, such as sperm, by distinguishing differences in DNA content. The cells are stained with a DNA specific dye by mixing the sperm with the dye and then electroporating them. The sperm can be maintained at temperatures that enhance sperm viability, typically equal to or less than <NUM>° C. The sperm are then passed before an excitation light source causing the stained DNA to fluoresce, and then passed through means for detecting the fluorescence and a means for cell sorting, wherein the cells are sorted by DNA content, and the sorted sperm collected. The methods and apparatus are appropriate for mammalian sperm sorting, such as those from bovine, swine, rabbit, alpaca, horse, dog, cat, ferret, rat, mouse and buffalo. Both membrane permeant and impermeant dyes can be used. Useful dyes include those from the SYTOX blue, orange and green series, cyanine dimers and monomers, POPO-<NUM>, BOBO-<NUM>, YOYO-<NUM>, TOTO-<NUM>, JOJO-<NUM>, POPO-<NUM>, LOLO-<NUM>, BOBO-<NUM>, YOYO-<NUM>, TOTO-<NUM>, PO-PRO-<NUM>, BO-PRO-<NUM>, YO-PRO-<NUM>, TO-PRO-<NUM>, JO-PRO-<NUM>, PO-PRO-<NUM>, LO-PRO-<NUM>, BO-PRO-<NUM>, YO-PRO-<NUM>, TO-PRO-<NUM>, TO-PRO-<NUM>, acridine homodimer, <NUM>-amino actinomycin D, ethidium bromide, ethidium homodimer-<NUM>, ethidium homodimer-<NUM>, ethidium nonazide, nuclear yellow, propidium iodide. Other useful dyes include those from SYTO <NUM> blue, green, orange and red fluorescent dyes, Hoechst dyes and dihydroethidium. To enhance the signal, nanoparticles, such as quantum dots and metallic nanoparticles, can be introduced. The particles can be tagged with targeting molecules. Sorting efficiency can be greater than <NUM>%, while sperm viability rates are greater than <NUM>%, typically greater than <NUM>%. Alternatively, instead of electroporating the cells, the dye is introduced into the cells by osmotic gradients. Cells are first incubated in hypertonic conditions, and then transferred to hypotonic conditions; the DNA-staining dye can be added to either, or both, hypertonic and hypotonic solutions. After dying the cells, they are ready to be sorted or further processed. In other aspects, the systems and methods of the invention may involve detecting differences in DNA content without the use of DNA stains, for example by <CIT>, <CIT>; and <CIT>. In another aspect, the systems and methods of the invention may involve detecting characteristics of particles using other means of analysis, including for example side-scatter, forward scatter, or back-scatter of light, or a live/dead stain (such as propidium iodide or other viability stains).

In another aspect, the systems and methods of the present invention may involve establishing or modifying a threshold or gate. A threshold generally refers to a value, typically related to a particular characteristic, that can be used to classify individual events. For example, an intensity threshold can be set at a value that will permit discrimination of fluorescence as indicative of live cells (e.g. above the threshold value) or dead cells (below the threshold value). Similarly, a gate refers to a set of points or vertices that bound a region in a two-dimensional graphical representation of detected characteristics. For example, a gate may refer to a set of four or more points that are connected to form a polygon around a subpopulation. A gate can be used to define or discriminate a particular subpopulation. The individual points or vertices that make up a gate can be discrimination values, calculated from event data. On a scatter plot, each point or vertex of a gate has an X and a Y coordinate. The value of these coordinates can be determined by calculation based on complied event data, and/or by values contained in the parameter set. The gate can then be used to classify individual events as to whether they fall inside or outside of the gate. For example, the gate may be established to be inclusive of a particular cell type, based on the presence of fluorescent markers bound to two cell surface markers; cells that have both markers will fall within the gate, while any cells lacking one or both markers will fall outside the gate.

In one aspect, a system for optimized particle discrimination and sorting is provided. In one aspect, the system includes a sample delivery apparatus. A mixture of particles is delivered to the cytometric system by a sample delivery system. As will be appreciated by a person of skill in the art, sample delivery may be accomplished using pumps, including syringe pumps, peristaltic pumps, and or an increase of pressure differential between the sample and the system. Generally, sample delivery systems are capable of either discretely or continuous sampling a mixture of particles in liquid suspension, and moving the mixture of particles to the cytometric apparatus. In some applications, the sample delivery system may also involve mixing the mixture of particles with another fluid, and/or delivery of a buffer or sheath fluid. The sperm sample to be sorted may be a freshly collected sample from a source animal, such as bovine, equine, porcine, or other mammalian source, or a thawed, previously cryopreserved sample. Moreover, the sample may be a single ejaculate, multiple pooled ejaculates from the same mammal, or multiple pooled ejaculates from two or more animals.

Various collection methods are known and include the gloved-hand method, use of an artificial vagina, and electro-ejaculation. The sperm are preferably collected or quickly transferred into an insulated container to avoid a rapid temperature change from physiological temperatures (typically about <NUM> to about <NUM>). The ejaculate typically contains about <NUM> to <NUM> billion sperm per milliliter, depending upon the species and particular animal.

Regardless of the method of collection, an aliquot may be drawn from the sperm sample and evaluated for various characteristics, such as for example, sperm concentration, sperm motility, sperm progressive motility, sample pH, sperm membrane integrity, and sperm morphology. This data may be obtained by examination of the sperm using, for example, the Hamilton-Thorn Motility Analyzer (IVOS), according to standard and well known procedures (see, for example, <NPL>; and <CIT> and <CIT>).

In another aspect, the systems of the present invention include a cytometry apparatus. The cytometry apparatus detects characteristics of particles as events, and provides signals corresponding to events to a processor. According to certain aspects, a flow cytometry apparatus includes: a flow chamber configured to direct a fluid stream including sample particles through a particle interrogation location; a laser configured to emit electromagnetic radiation along a beam path to the particle interrogation location; a detector configured to receive electromagnetic radiation from the interrogation location; and output a time-varying analog signal indicative of an intensity of the received electromagnetic radiation; at least one amplifier configured to amplify the time-varying analog signal; an analog-to-digital converter configured to receive the amplified time-varying analog signal and produce a corresponding digitized output signal. In further aspects, the detector may be a photomultiplier tube (PMT) or an avalanche photodiode (APD). The cytometer may further include a temperature sensor configured to sense a temperature and generate a corresponding temperature signal encoding temperature data, wherein the voltage adjustment circuitry is further configured to adjust the voltage on the feedback loop based at least in part on the temperature data and the voltage measured between the filter and the avalanche photodiode. The voltage adjustment circuitry may further be configured to adjust the voltage on the feedback loop based at least in part on at least one characteristic of the avalanche photodiode, the temperature data, and the voltage measured between the filter and the avalanche photodiode. The voltage adjustment circuitry may further be configured to adjust the voltage on the feedback loop based at least in part on at least one characteristic of the avalanche photodiode and the voltage measured between the filter and the avalanche photodiode. The voltage adjustment circuitry may include: an analog-to-digital converter configured to convert the voltage measured between the filter and the avalanche photodiode into a digital measured signal encoding measured voltage data; a processor configured to process at least the measured voltage data to generate a digital adjustment signal; and a digital-to-analog converter configured to convert the digital adjustment signal to an adjustment voltage, wherein the adjustment voltage influences the voltage on the feedback loop. The processor may be configured to process at least temperature data and the measured voltage data to generate the digital adjustment signal. The processor may be configured to process at least temperature data, data corresponding to at least one characteristic of the avalanche photodiode, and the measured voltage data to generate the digital adjustment signal. The at least one characteristic of the avalanche photodiode may include at least one of a breakdown voltage and a reverse bias voltage corresponding to a predetermined optical gain. The cytometer may further include: a first amplifier configured to amplify a voltage at an anode of the avalanche photodiode to form a first amplified voltage; and a second amplifier configured to amplify the first amplified voltage to generate a second amplified voltage. The power supply may include a DC/DC power supply. The avalanche photodiode may be: arranged to receive an amount of fluorescent light emitted by each of a plurality of particles; the amount of received fluorescent light may vary based at least in part upon a relative amount of at least one particle differentiation characteristic present in each of the plurality of particles; and the avalanche photodiode may be configured to convert the amount of received fluorescent light into at least one signal which varies based upon the amount of received fluorescent light.

A stream of particles may pass through a flow chamber, such that they may be single-file in certain strategic locations. The flow chamber may direct a fluid stream including the mixture of particles through a particle interrogation location. The mixture of particles may have been previously stained with a dye, such as a DNA-intercalating dye. Such a dye may fluoresce (or cause fluorescence) as it generates responsive light in response to being exposed to a light (or electromagnetic radiation) source. As the particles pass one-by-one, they may be exposed to a beam of electromagnetic radiation (for example, light of a given wavelength) generated by a laser and emitted along a beam path (and optionally associated optics) to the particle interrogation location. Such associated optics may include lenses, filters, or the like. The exposure of the particles (including particles stained with a dye) to the electromagnetic radiation may cause a responsive event, including light scattering, absorption, and/or fluorescence emitted by stained particles. In some aspects, the amount of fluorescently generated light may vary by a detectable degree, depending on whether sperm cell particles bear an X chromosome or Y chromosome.

The fluorescently generated light may be received by optics (for example, lenses, filters, or the like), and it may be focused onto an active area of a photodetector. According to certain inventive techniques, the photodetector may include an APD or PMT. The photodetector may generate an output signal that corresponds (varies linearly or non-linearly) to the amount of electromagnetic radiation (for example, light of a given wavelength) that it receives from the interrogation location. The photodetector output signal may include a time-varying analog signal indicative of an intensity of the received electromagnetic radiation. This photodetector output signal may ultimately be communicated to a processor (which may include one processor or a plurality of processors that control a portion of the operation or the entire operation of the flow cytometer or the sorting apparatus). The photodetector output signal may be amplified by at least one amplifier (or two or more amplifiers in series) before it is communicated to the processor. Furthermore, the photodetector output signal (for example, as amplified) may be digitized before being communicated to the processor.

In a further aspect, the system may include a processor that receives a signal, and generates even data. The processor may further compile event data into one or more data sets. The processor may further distribute event data in the data set, makes discrimination value calculation; classifies events based on discrimination value and makes sort decision; and/or provide instruction to sorting apparatus. The processor may comprise a single component or may comprise multiple components. The processor is configured to analyze the digitized output signal received from the flow cytometry apparatus. In general, electrical signals from the cytometer detector (i.e. PMT or APD) are converted to digital information by an A/D converter which supplies the corresponding digital information to the microprocessor. In response to the information, the microprocessor controls a sorting system.

The electrical signals output from the photodetector of the cytometry system are time-varying analog voltage signals indicative of the amplitude of the emitted fluorescence at any instant in time generated by each particle as it is illuminated by the laser beam. Thus, the analog signals (also referred to as analog output) are in the shape of time-varying waveform pulses. In general, a waveform pulse is defined as a waveform or a portion of a waveform containing one or more pulses or some portion of a pulse. Thus, the amplitude of each waveform pulse at any instant in time represents the relative rate of photon emission of each cell at that instant in time as the cell passes through the laser beam. In one embodiment, X chromosome bovine sperm cells have a higher DNA content than Y chromosome bovine sperm cells (e.g., about <NUM>%). As a result, live X cells labeled with a fluorescent stain as noted above will produce a different waveform pulse <NUM> than pulses from any other labeled cells. By analyzing the pulses as noted below (see Signal Processing, Slit Scanning, and Critical Slope Difference), each cell can be identified as an X cell or not identified as an X cell (~X). In general, as used herein, X cells refers to live X cells, Y cells refers to live Y cells and ~X cells refers to the combination of live Y cells and cells which otherwise produce a detectable fluorescence emission but which cannot be identified with a reasonable probability as being live X cells.

The timing of each waveform pulse indicates the position of each cell in the stream. Since the rate at which the particles are being delivered through cytometer remains constant, and since the distance between the detector and the sorting apparatus is known, the position of each particle known. Thus, the microprocessor can calculate the instant at which a sorting event occurs (i.e. when the ablation laser is fired). Since the microprocessor receives event data for each particle, including the characteristics for the particle, the microprocessor may keep track of (or enumerate) the number of particles that can be classified in each subpopulation. Depending on the parameter set and sorting strategy implemented by the control apparatus, see below, the microprocessor determines how the particles it the mixture of particles are sorted.

In another aspect, the system includes a control, in the form of a microprocessor (or other digital or analog control and/or processor, or combinations thereof) that controls the discrimination of particles within the mixture of cells. In a further aspect, this control apparatus receives information from the cytometer apparatus, including measured characteristic data for particles in the mixture of particles. The control further includes the information present in the parameter set. The control is capable, using these sources of information, of distinguishing the values of measured characteristics for sub-populations in a set of particles; assigning a critical range of values to two or more sub-populations based on the distinguished values of the measured characteristic; measuring a second set of particles in the mixture of particles, and distinguishing the values of the measured characteristics for sub-populations in the second set of particles; updating critical range of values assigned for the subpopulations using the values of the measured characteristic for the subpopulations distinguished for the second set of particles; and classifying particles in the mixture according to a sub-population classification, using the updated critical range of values. The control provides output signals to control the sorting system based on the classification. The control may be implemented within a single microprocessor. Alternatively, some or all functions may be integrated into one or more processors. In addition, the processing may be implemented by an analog circuit or a combination of analog and digital circuitry.

The control may provide output signals to other parts of the cytometry system. Further, the control may be adapted to process information and provide output signals in real time. Broadly speaking, the term "real time" refers to operations in which the operation of the control apparatus matches the human perception of time or those in which the rate of the operation of the processor or processors comprising the control matches the rate of relevant physical or external processes. In one context, the term "real time" can indicate that the system reacts to events before the events become obsolete.

In another aspect, the systems of the present invention include a sorting apparatus. The sorting apparatus carries out a sorting operation on the particles in the mixture of particles, according to the instructions received from the control apparatus. The sorting apparatus can be any type of sorter known to a person of skill in the art, including for example a droplet sorter, a mechanical sorter, a laser ablation ("zapper") system, or using any other technique known to a person of skill in the art]. In one embodiment, the sorting system comprises a focused energy apparatus, as described in <CIT>. A focused energy apparatus is used to provide focused energy pulses to the particles. The focused energy apparatus may be a thermal, electrical, optical, or electromagnetic device, which would have a desired wavelength, and would deliver high peak power with a very high repetition rate (or pulse frequency), to the target particles. In one embodiment, the focused energy apparatus is triggered a predetermined time (i.e., milliseconds) after activation by the controller (the timing being set based upon the traveling speed of particles through the channel) and issues a pulse to the selected or targeted (i.e., unwanted) particle.

Examples of pulsed lasers include mode-locked, Q-switch, as well as those lasers using both mode-locking and Q-switch techniques. For example, a focused energy device such as an Avia <NUM>-<NUM>-<NUM> (made by Coherent, Inc. , Santa Clara, Calif. ), or the Explorer XP lasers Q-switch laser from Spectra-Physics Inc. , is capable of operating in a pulse-on-demand mode, and can deliver <NUM> ns energy pulses or less, at a rate of over <NUM> pulses per second, to the target objects. In one embodiment, pulse energy levels of <NUM>-<NUM>µJ are used, and in a preferable embodiment, a Q-switch laser in pulse-on-demand mode is used to deliver an average pulse energy of <NUM>µJ with a range for individual pulses of <NUM>µJ to <NUM>µJ. In one embodiment, the pulse width ranges from <NUM> nanoseconds to <NUM> microsecond, and preferably, is in a range of <NUM>-<NUM> nanoseconds. However, one of ordinary skill in the art would know that any high power laser existing now, or later developed, with the appropriate high energy pulses and pulse frequency, would be suitable for the present invention in order to achieve the desired target accuracy and/or effect.

In one embodiment, the need for a tight action region in order to deliver the pulsed energy from the focused energy apparatus to target particles or a surrounding region thereof, is important to minimize the potential impact of delivering the energy outside of the targeted particles or region, to otherwise unselected, or non-targeted particles. For example, a focused energy apparatus such as the Explorer XP <NUM>-<NUM> Q-switch laser, is capable of delivering <<NUM>% rms, providing high pulse-to-pulse stability when fired at regular uniform intervals. However, for flow cytometric analysis and action systems where particles or cells enter the action region in non-uniform intervals, additional measures are employed to deliver uniform pulse energy to impinge only the targeted particles or cells, or the surrounding region thereof. Such measures include matching laser performance parameters such as pulse length and peak power levels, to enable the system of the present invention to achieve a desired target accuracy (i.e., in one embodiment, photodamage or kill rate of <NUM>% or higher hit rate on target particles). In addition, further tuning the pulse-on-demand operation and performance of the laser to deliver extremely high pulse-to-pulse stability when fired at non-uniform intervals, greatly reduces the spatial variability in the area impacted by the pulse. Thus, by reducing pulse-to-pulse variability in the focused energy device, the unintended action, damage or destruction to non-target particles or cells, is greatly reduced, achieving, for example, an <NUM>% or higher rate of viability for live, non-target particles or cells.

The focused energy apparatus can be set to damage, alter, disable, kill or destroy the targeted or unwanted particle or cell in the sample fluid with a pulse, or to activate one of several mechanisms in the particle or cell, such that cell damage or death ensues. However, depending on the desired arrangement, the targeted or selected particle may be wanted objects, in which case the focused energy apparatus is not activated or triggered, or the targeted or selected particles may be unwanted particles, where the focused energy apparatus is activated act upon the particles, such as to damage, alter, disable, kill or destroy the selected, unwanted particles. However, these are not the only embodiments, and the various embodiments are discussed further below.

In one embodiment, when the selected particle or cell is damaged, altered, disabled, killed, or destroyed by the focused energy device, the particle or cell continues to flow through the channel, and into container, along with any non-targeted particles or cells. Any sheath or buffer fluids also proceed in laminar flow through output channels.

Accordingly, in one embodiment, the present methods and systems are capable of producing a discriminated product of particles or cells in a container, including a high viability of non-target or wanted particles or cells, and a high percentage of photodamaged, altered, disabled, destroyed, or dead target particles or cells.

In another aspect, a method is provided for identifying subpopulations within a mixture of particles. The mixture of particles may contain two or more sub-populations. The subpopulations may be distinguishable from one another by a characteristic, or more than one characteristics. The method comprises measuring a characteristic of a first set of particles in the mixture of particles. The method further comprises distinguishing the values of the measured characteristics for sub-populations in the set of particles. In one aspect, values of the measured characteristics may be distinguished for two or more sub-populations in the set of particles. In another aspect, values of the measured characteristics may be distinguished for each of the sub-populations in the set of particles. The method further comprises assigning a critical range of values to two or more sub-populations based on the distinguished values of the measured characteristic. The assigning of a critical range can be by identifying (or making a classification of) a sub-population in relation to other subpopulations based on the distinguished values of the measured characteristic for each of the subpopulations being compared. Subsequent to the assignment of a critical range of values, the method further comprises measuring a second set of particles in the mixture of particles, and distinguishing the values of the measured characteristics for sub-populations in the second set of particles. The critical range of values assigned for the subpopulations is then updated using the values of the measured characteristic for the subpopulations distinguished for the second set of particles. The method further comprises classifying particles in the mixture according to a sub-population classification, using the updated critical range of values.

In one aspect, the methods of the present invention are performed on a mixture of particles in order to identify, discriminate, distinguish, or sort the particles within the mixture. The mixture of particles may contain two or more subpopulations. The subpopulations may be distinguishable from one another by a characteristic, or more than one characteristics. The particles may be cells. The cells may be of a single type, or may be a mixture of multiple cell types, such as a mixture of cells derived from a tissue, or present in blood or blood parts. In a further aspect, the cells may be sperm cells. The sperm cells may be from any type of animal. In a further aspect, the sperm cells are from non-human mammals. In a further aspect, the sperm cells are from livestock animals. Livestock animals can include, but are not limited to fowl, ungulates, ruminants. In a further aspect, the sperm cells are ovine, caprine, equine, porcine, or bovine. Bovine sperm cells can include sperm cells from domestic cattle, bison, African buffalo, water buffalo, or yak.

In another aspect, characteristics of particles or sets of particles are detected and measured. Characteristic can be any aspect of a particle that can be detected. Characteristics may include aspects such as size, shape, permeability, integrity, content, vitality, mortality, metabolic status, and surface markers. In a further aspect, the characteristics may include DNA content. DNA content, particularly in haploid cells such as sperm cells or ova, is an indicator of the presence of one of the two sex chromosomes. The X-chromosome is larger than the Y chromosome, and therefore a haploid cell that contains an X-chromosome will contain more total DNA than a gamete that contains a Y-chromosome. The characteristic can be detected using a variety of methods knows to a person of skill in the art, including light scatter, interference, absorbance, or detection of fluorescence. For example, the characteristic of DNA content of a cell can be detected by staining the cell with a DNA-binding fluorescent dye, and detecting the fluorescence of the dye. In certain respect, characteristics of particles are measured using a flow cytometer. The use of flow cytometers for detection and measurement of characteristics is known in the art, but in general cytometers operate by converting the incidence of a photon with a detector into an electrical signal. He electrical signal from the detector therefore corresponds to the electromagnetic radiation from a particle (in the form of photons emitted by or scattered from the particle) upon exposure to a source of electromagnetic radiation. Frequently, fluorescent molecules are utilized to specifically label a particular aspect of the particle, and the cytometer detects photons of a specific wavelength emitted by the particle in response to exposure of the particle to certain electromagnetic radiation. The electromagnetic radiation from the particle, indicative of a characteristic, is detected by a detector within the cytometer apparatus. The detector is usually either a photomultiplier tube (PMT) or an avalanche photodiode (PMT) as described in <CIT>. The detector converts photons into an electrical signal that can be transmitted to the processing components of the cytometer, resulting in the assignment of values to the detected characteristic. Distinguishing values of the measured characteristics, based on the valuation derived from the cytometer, may further involve sorting and comparing values for particles or sets of particles to elucidate the features of subpopulations within the mixture of particles. This can be achieved, for example, by determining whether the values, when represented graphically, form groups or peaks. The identity of the subpopulations can then be ascertained based on the relationship of any groups or peaks that are thus elucidated. In some aspects, the distinguishing can be through finding discrete groupings of events based on the measured characteristic or characteristics. The measured characteristics can be represented in a feature space. A feature space, for example, can be a graphical representation of or more measured characteristics of the particles. In one exemplary embodiment, the feature space can be a histogram of fluorescent intensities, or a scatter plot of fluorescent intensities and total fluorescence, wherein the fluorescence is indicative of the amount of DNA present in a cell. In some aspects, the distinguishing can be discrimination between two sub-populations, discrimination of a single subpopulation amongst several, or discrimination of a subpopulation within a continuum.

In further aspects, event data is generated for particles by the cytometer apparatus. The event data is typically generated based on the detection of electromagnetic radiation emitted by, or scattered from, particles upon exposure to a source of electromagnetic radiation. The electromagnetic radiation is converted into electrical signals by a detector (usually either a PMT or and APD). The electrical signal is then converted into data by the processor components of the cytometer. In certain embodiments, the event data may be fluorescence intensity (i.e. the highest fluorescence value for an individual event corresponding to a single particle) and/or the total fluorescence (i.e. the integrated fluorescence values for the entire event corresponding to a single particle).

In certain aspects, the methods of the present invention involve discrete actions carried out on particular sets of particles. For example, the measuring a characteristic of particles in a mixture of particles and distinguishing of values of the measured characteristics can be performed on a first set of particles from a mixture of particles, and subsequently the measuring a characteristic of particles in a mixture of particles and distinguishing of values of the measured characteristics can be performed on a second set of particles from the same mixture of particles.

In a further aspect, the methods involve assigning a critical range of values to each of the sub-populations based on the previously distinguished values of the measured characteristics. In certain respects, the assigning of critical ranges is used to identify specific subpopulations within the mixture of particles. For example, a critical range may be assigned to a set of particles to identify that set of particles, based on the relationship of the peak formed by the values for those particles represented graphically, in relation to the peak of values for other particles. The assignment of a critical range of values may include the calculation of a discrimination value. The discrimination value can be a threshold discrimination value (single value or two values) or gate threshold values (e.g. three or more values). The calculation may use one or more values from the parameter set. In certain embodiments, the calculation of a discrimination value involves identifying at least one critical value for a first characteristic from the distribution of event data for the first set of particles; obtaining the values for a second characteristic associated with the critical value for the first characteristic; producing a distribution of the values for the second characteristic; identifying at least one critical value for the second characteristic from the distribution of values for the second characteristic; co-locating the critical values for the first and second characteristics in a feature space; and determining the at least one discrimination values from the critical values co-located in the feature space. In further aspects, calculation of a discrimination value may further involve adjusting the critical values to include event data from a second set of particles. In further aspects, the critical values comprise one or more peak characteristic values for a first sub-population, and one or more peak characteristic values for a second subpopulation. The peak characteristic values comprise peak fluorescence intensity and peak total fluorescence. In further aspects, the critical values may also a trough value between the peak characteristic values for the first and second subpopulations.

In a further aspect, the methods may include measuring the characteristics of and distinguishing the values of the measured characteristics for each of the sub-populations in the second set of particles in the mixture of particles, which occurs subsequent to the assigning a critical range of values for the first set of particles. This measuring and distinguishing on the second set of particles is carried out in order to determine whether the assignment of the critical range of values to the sub-populations is accurate, and if not, to update the values used to make that assignment. In some aspect, the values for the second set of particles is compared to those for the first set of particles, and if they are not different, the critical range of values assigned based on the first set of particles is used. If there is a difference in the values for the first set of particles and the second set of particles, the critical range of values is updated using the values for the second set of particles. based on the distinguishing of the values of the measured characteristics for each of the sub-populations in the second set of particles. Typically, a certain number of particles, or the number of particles that can be measured in a set amount of time, dictates the composition of each set of particles, and therefore how often the critical range of values is updated. The number of particles that comprises a set and/;or time value is part of the parameter set.

In a further aspect, individual particles in the mixture of particles are assigned a classification based on the assigned critical range of values. This can be based on a critical range of values that is or is not updated as a result of a new set of particles being measured. This classification of particles is derived from the relationship of the subpopulations to one another. Individual particles in the mixture can be classified as belonging to a subpopulation according to this assignment of critical values, while as the same time these same particles that are classified are part of a second set of particles used to update the critical range of values.

In a further aspect, the methods of the present invention provide for identifying subpopulations within a mixture of particles by defining gates to discriminate one or more subpopulations of particles within a mixture. As the particles within the mixture are processed by flow cytometry, the particles are detected by the detector apparatus of the flow cytometer, generating a detector event. Each detector event can be an aggregation of values, including, for example, values for intensity and area of a signal. A computer is used to process the event data. Event data is accumulated until the data for a sufficient number of events has been compiled to permit the calculation of one or more discrimination values. A parameter set, that is a set of values that affect certain details of all discrimination value calculations, are provided before any discrimination values are calculated. When sufficient event data has been compiled, a discrimination value is calculated, and the process is repeated with a new set of event data. Each set of event data is the previous set of event data to determine whether the discrimination value calculated for the subsequent set of event data is significantly different from the previous discrimination value calculated from the previous set of event data. If it is not significantly different, data resulting from the previous discrimination value calculation, if available, may be averaged with data from the subsequent discrimination value to improve consistency of location and shape between consecutively-calculated discrimination values. If the discrimination value is significantly different, data for the previously calculated discrimination value is discarded.

In certain aspects of the methods, a mixture of particles is provided. The composition of the mixture can vary according to the application. The mixture may include particles and medium. In certain embodiments, the mixture of particles is a sperm samples. The sperm cell may be present with components of raw ejaculate, with other media, and/or extender. The mixture of particles is delivered to a flow cytometry system.

In certain aspects, a parameter set is provided. The parameter set is provided to the processor for calculation of one or more discrimination values. The parameter set includes a set of values that affect certain details of all discrimination value calculations. In some respects, the parameter set includes values that are used to calculate a threshold value. In other aspects, the parameter set includes values that are used to calculate one or more points or vertices of gate. In further aspects, the parameter set includes the frequency at which calculation of a discrimination value is to occur, and/or the quantum of event data that triggers calculation of a discrimination value.

In certain aspects, the characteristics of particles in the mixture are detected. This detection is performed by the cytometry apparatus. The cytometer detects characteristics by detecting aspects of individual particles. The detection of characteristics of an individual cell is considered a single event. In some cases, the particles may arrive at the detector as clumps of particles, and thus the detection will proceed for the clumped cells as a single event. These aspects can include size and shape of the particle, for example by detecting light scatter, and particle composition, for example by detecting the presence of a fluorescent marker on the surface of the particle or within the particle. In certain respects, the particle aspect being detected may be a fluorescent dye. In further respects, the particle may be a cell, and the dye may be a fluorescent DNA dye. One or more characteristic may be detected for each particle, and a single event may include one or more detected characteristics. The detector of the cytometry apparatus generates a signal that corresponds to the detected characteristic or characteristics for each event and provides that signal to a processor (e.g. a CPU).

Compiled event data is distributed. Occurrence of data distribution may depend on one or more values from the parameter set - frequency or number of events. In some respects, the distribution may be a sorting of events by event area. In other respects, the distribution may be sorting of events by intensity. In further respects, the distribution may be sorting of events by intensity and area.

In a further aspect the classification of events is provided to the sorting apparatus, which implements the sort/no-sort decision.

The discrimination value calculated for a set of compiled events is compared to the discrimination value calculated for the previous set of events. Repeat steps to generate a new discrimination value based on a new compiled set of event data. If different, discard previous; if not different, average event data. This can be done by comparing the calculated discrimination values directly, or by comparing the data in the two compiled data sets.

In a further aspect, the methods of the present invention can be carried out in the following manner:.

In a further aspect, the process further comprises inventive techniques for finding a drift-corrected intensity [expansion of steps <NUM>-<NUM> above].

In certain aspects, the present invention provides sorted particle products produced by the systems and methods described herein. These sorted products may be of a higher and/or more consistent quality. In certain aspects, the sorted particle products of the present invention are sexed sperm cell samples. The sexed sperm cell samples may be predominantly X-chromosome bearing sperm cells, or predominantly Y-chromosome bearing sperm cells. The sexed semen samples may be at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% at least <NUM>% at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>% (or any percentage within the range of <NUM>-<NUM>%) X-chromosome bearing or Y-chromosome bearing sperm cells. The sexed sperm cell samples may comprise a salt-based media formulation, optionally including any number of beneficial ingredients, including but not limited to, cryopreservation agents (such as glycerol), anti-oxidants, extenders, proteins, vitamins, and sugars. The sexed sperm cell samples may be suitable for fertilization, including by artificial insemination (AI) or in vitro fertilization (IVF).

Discrimination of Y chromosome-bearing sperm cells using programmed optimized gating system.

For certain implementations of semen sexing, the mixture of cells present in raw ejaculate (along with any dead cells and clumps of cells) is enriched for either live X chromosome bearing sperm cells or Y chromosome bearing sperm cells. For dairy applications, X chromosome bearing sperm cells are preferred to produce pregnancies that will result in female offspring. The enrichment is usually performed either using droplet sorting or by laser ablation. In laser ablation applications, a "kill" laser attempts to kill all cells that do not have the desired characteristic (i.e. any cell that is not a live X chromosome bearing sperm cell). In the present system, the cells are labeled with a DNA-intercalating fluorescent dye, and the fluorescence emitted by each cell is detected as a measure of the total DNA content. The fluorescent signal produced by each cell (or clump of cells) is detected by an avalanche photodiode and recorded as an event. X chromosome bearing sperm cells in cattle possess approximately <NUM>% more total DNA than Y chromosome bearing sperm cells, which translates into more fluorescence emitted by an X chromosome bearing sperm cell. This is detected as a larger fluorescence intensity (peak) for an event corresponding to an X chromosome bearing sperm cell, compared to an even corresponding to a Y chromosome bearing sperm cell. The area of the fluorescence signal for each event is also determined. The fluorescence signal area is an integration (i.e. the sum or total) of all the fluorescence from an event over the amount of time over which that event is detected. Each event can be represented graphically on a chart of intensity (peak) and area, and when the plots of many events are compiled, subpopulation of cells can be identified. For example, a subpopulation of X-chromosome bearing sperm cells are identifiable in relation to the Y chromosome bearing sperm cell subpopulation by its relatively higher intensity, and as individual cells based on the area (a doublet of cells will have a higher intensity and/or a larger area). The kill laser is triggered to fire at any event for which the event data produces a result that falls outside a certain region on the graph (i.e. the region containing the X-chromosome bearing sperm cells). The bounds of this region on the graph are the gate. In previous iterations of the system, a software operator created a gate by clicking on locations in the peak-intensity graph. The polygon describing the gate was created by sequentially selecting vertices until the polygon was completed. If the polygon was successfully described (i.e. it comprised at least three vertices) the coordinates of the gate vertices were sent to the GPP and from there to the DSP, to control the kill laser such that the kill laser was triggered by an even that was detected as falling outside the gate.

It was observed that different operators drew gates in different ways resulting in an unknown amount of variability in specific desired results, whether it be increased purity or increased production of sexed semen samples. Further, previous work has demonstrated that a gate which ascribes a subpopulation at the beginning of a run may not ascribe that same population at the end of the run, potentially due to several factors. Thus, there were three issues in the previous systems that needed to be resolved: lack of control over shape and location of gate creation, leading to product inconsistency and suboptimal collection conditions; inadequate centering of the gate around the female peak as event attributes drift during collection; and attendance by an operator to establish and maintain the fit of the gate to the X chromosome bearing sperm cell population.

To solve these issues, an algorithm was developed to set the gate vertices to draw the gate based on the known relationship between the X chromosome bearing sperm cell population and the Y chromosome bearing sperm cell population. Specifically, when compiling the events, the vertices of the gate were determined from the distribution of the events by use of an algorithm to perform the following operation. In the performance of the algorithm, the system uses a parameter set that includes: the frequency of the gate calculation and the maximum number of events whose data will be directly used for a gate calculation; the peak-to-trough (P2T) threshold, and the configuration settings for calculating the gate vertices. The algorithm is carried out as follows:.

A laser ablation semen sexing cytometer was adapted to implement the optimized sorting strategy set out in the above example. Semen from a <NUM>-year old Holstein bull was collected and prepared according to standard procedure. Briefly, collected semen was assessed for motility and the presence of abnormalities, and found to meet standards for processing. The sperm cell concentration was then determined, and the sample was stained with Hoechst <NUM> at <NUM> for <NUM> minutes prior to sexing. The sexing instrument includes a flow controller for delivery of the stained sperm cells and sheath fluid to a microfluidic chip. Sperm cell detection is carried out using a Vanguard™ mode-locked <NUM> laser (Spectra-Physics®), and fluorescence is detected using an avalanche photodiode. Unwanted Y-chromosome bearing sperm cells are ablated using an Explorer XP lasers Q-switch laser (Spectra-Physics®). Stained perm cells were processed over approximately <NUM> hours.

Discrimination of X- and Y-chromosome bearing sperm cells was carried out using an optimized gating strategy according to an embodiment of the present invention, as detailed in the above Example. Following sorting, the collected sample yielded approximately <NUM> million sperm cells. During operation, the values of the various gating parameters were monitored to observe the operation of the gating system. Extracted portions of the histograms of intensity values generated from events at two timepoints ten minutes apart during a single sample run were compared. Curves were fitted to the peaks as well as to the trough; mathematical analysis of the fitted curves yields the more precise peak and trough bin values shown above each graph. The precise peak and trough values were converted to actual intensity values by multiplying by the bin size, which is <NUM>. Thus, for example, the male peak intensity in the histogram on the left is <NUM> x <NUM> = <NUM>.

Comparison of the intensity values for the peaks at the two different time points shows a significant shift in the female peak intensity from <NUM> to <NUM> (<NUM> intensity units, or about <NUM>%) over ten minutes. The trough similarly shifts from <NUM> to <NUM> (<NUM> intensity units). The gate must shift a similar amount in order to maintain the correct relationship to the location of the female peak.

Since a gate is to be constructed around the female peak, similar histograms are constructed programmatically for the 'area' values of all events whose intensities are near the female peak intensities. Those histograms only show one peak. The analyzed histograms of the 'area' values placed the female area peaks at <NUM> at the first time point and at <NUM> at the second time point.

All the intensity coordinates of the gate shift left (lower) over the ten minutes between the two time points. The intensity coordinates of the gate vertices at the first time point average <NUM>. The corresponding average at the second time point (<NUM> minutes later) is <NUM>. This corresponds to an average shift downwards of <NUM> intensity units. This compares favorably with the shift downwards of the female peak intensity of <NUM> intensity units, and of the intensity at the trough's shift downwards of <NUM> intensity units. The gate has shifted left to maintain the relationship with the female peak.

The female peak's area coordinate also shifts downwards during the ten minutes. All the gate's area coordinates for the vertices shift downwards similarly.

Differences between the precise amounts of shifts in instantaneous peak coordinates versus gate vertex coordinates are primarily due to the use of trough-to-female-peak distance in calculating the vertices. Since the trough and female peak are independent statistical entities, the vertex coordinates can show a very slightly different shift between two timepoints than the female peak, although the average shifts of vertex coordinates and peak coordinates over time are equal.

Claim 1:
A method for identifying subpopulations within a mixture of particles (<NUM>), comprising:
measuring a characteristic of a first set of particles (<NUM>) in the mixture of particles (<NUM>) having two or more sub-populations, each sub-population being distinguishable by the characteristic;
distinguishing the values of the measured characteristics for each of the sub-populations in the mixture (<NUM>);
assigning a critical range of values (<NUM>) to each of the sub-populations based on the distinguishing;
subsequent to the assigning a critical range of values (<NUM>), measuring the characteristics of a second set of particles (<NUM>) in the mixture of particles (<NUM>);
subsequent to measuring the characteristics of a second set of particles (<NUM>) in the mixture of particles (<NUM>), distinguishing the values of the measured characteristics for each of the sub-populations in the second set of particles (<NUM>);
updating the critical range of values (<NUM>) based on the distinguishing of the values of the measured characteristics for each of the sub-populations in the second set of particles (<NUM>) in real time; and
assigning a sub-population classification to particles in the mixture (<NUM>) based on the updated critical range of values (<NUM>).