SYSTEM FOR PERFORMING ANALYSIS AND SELECTION OF SPERM AND METHOD FOR CLASSIFICATION OF SPERM CELLS

专利摘要:
the invention provides a system (1) to perform sperm analysis and selection based on sperm cell morphology of sperm cells (6) in a fluid (5), the system (1) comprising: (i) a flow channel of fluid (2) for carrying said fluid (5), the fluid flow channel (2) comprising an inlet (10), an analysis zone (40) configured downstream of said inlet (10) and comprising a first pair of electrodes (41) comprising a first intra-electrode distance (dl), a classification zone (50) configured downstream of said analysis zone (40) and comprising a classification device (51), and outlets (80, 90 , ...) configured downstream of said classification zone (50); (ii) an electrical source (140) configured to provide an electrical signal to the first pair of electrodes (41); (iii) a measuring device (150) coupled in a functional way to the first pair of electrodes (41) and configured to measure a first impedance as a function of fluid time (5) between the first pair of electrodes, and to provide data time-dependent impedance; wherein the sorting device (51) is configured to classify sperm cells (6) by directing the sperm cell (6) in the sorting zone (50) to one of the outputs (80, 90, ...) based on a comparison in a time-dependent impedance data comparison stage with predefined reference data.
公开号:BR112018006145B1
申请号:R112018006145-0
申请日:2016-09-30
公开日:2021-04-13
发明作者:Bjorn de Wagenaar；Loes Irene Segerink；Wouter Olthuis；Adrianus Joseph Sprenkels；Albert Berg Van Den
申请人:Semen Refinement B.V.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The invention relates to a system for classifying sperm cells as well as a method for classifying sperm cells. The invention also relates to a sperm product. BACKGROUND OF THE INVENTION
[002] The maintenance of sperm morphology and motility to increase the success rate of artificial insemination is known in the art, and is, for example, described in WO2004053465A2. In this document, a method and device for testing sperm motility in a forward direction and active sperm density in a semen sample are described. The device includes a microfluidic structure having a sample reservoir, a downstream collection region and a microchannel that extends between them. The microchannel is sized to restrict sample sperm to one-way movement within the channel, so that sperm in a semen sample placed in the sample reservoir between and migrate along the microchannel towards and from the collection region. Also included is a detector to detect the presence of marked sperm in the microchannel or collection region, and an electronic component unit operatively connected to the detector to (i) receive detector signals, (ii) based on the received detector signals , determination of sperm motility and density in the sperm sample, and (iii) display of information related to sperm motility and density.
[003] EP2508253A1 describes a channel device including a nano-sized channel through which a single molecule flows, at least one pair of electrodes arranged close to the nano-sized channel, and an AC power source that applies an AC voltage to the electrodes. This channel device is useful for identifying molecules one by one. In addition, a channel device is described, including a nano-sized channel through which a single molecule flows, a branching part, and a plurality of branching channels, in which (i) a pair of electrodes is arranged close to the channel nano-sized, in order to squeeze the nano-sized channel between the electrodes, or (ii) one electrode from the pair of electrodes is located next to the nano-sized channel, while the other is disposed close to the branching channels. This channel device is useful for classifying single molecule. The channel device achieves identification or classification with an accuracy of 100% in principle. Sample treatment equipment, according to the inventors, includes a channel device, a measuring section, and an arithmetic processing section. The measurement section applies a voltage (DC or AC) between the electrodes of a pair of electrodes installed in the nano-sized channel, and measures an electrical signal when the single molecule passes between the electrodes to identify the molecule.
[004] EP2211164 A1 describes that the electrical properties of particle solutions can be investigated in a single particle when using microfluidic channels. The impedance can be measured by the channel using at least one pair of conductive electrodes, at least one electrode of a pair being a fingered electrode having a plurality of fingers. The pattern of the fingered electrodes creates a longer and more complicated measurement signal form which leads to a significant improvement in the measurement sensitivity. One application for the proposed technology is to significantly improve the measurement sensitivity of impedance measurements in blood cells, leading to better differentiation between different types of leukocytes. Better measurement sensitivity also allows for the measurement of smaller particles and greater production.
[005] The document US2005 / 0118705 A1 describes equipment and methods to perform microanalysis of particles using a microelectric-mechanical system chip (MEMS) to electrically interrogate the particles. The MEMS chip is typically manufactured using known lithographic micromachining techniques, used, for example, in the semiconductor industry. A substrate carries a plurality of microelectrodes arranged in a detection zone and spaced along an axis of a microchannel. The microchannel is dimensioned in cross section to make the particles charged by a fluid move beyond the electrodes in a single process. Impedance is measured between one or more pairs of electrodes to determine the presence of a particle in the detection zone.
[006] US2013 / 0256197 A1 describes a flow channel device that includes a flow channel in which a fluid containing a particle flows, a plurality of branch channels of the flow channel, and an electrode unit. The electrode unit includes a first electrode having a first area and a second electrode having a second area different from the first area and is configured to form a guide electric field in the flow channel, which guides the particle to a predetermined branch channel of the plurality of branching channels. The second electrode is opposite the first electrode, so that the flow channel is pressed between the first electrode and the second electrode.
[007] Document US2014 / 0248621 A1 describes microfluidic devices and methods that use cells, such as neoplastic cells, stem cells, blood cells for pre-processing, classification for various bio-diagnostic tests or therapeutic applications. Microfluidic electrical detection, such as measuring potential or current or field phenomenon, such as immiscible fluids, inert fluids are used as the basis for cellular and molecular processing (eg characterization, classification, isolation, processing, amplification) of different chemical particles, compositions or biospecies (for example, different cells, cells containing different substances, different particles, different biochemical compositions, proteins, enzymes, etc.). Specifically, the document describes few classification schemes for stem cells, whole blood and circulating neoplastic cells and also serum extraction from whole blood.
[008] Segerink et al. describes in “On-chip determination of spermatozoa concentration using electrical impedance measurements” Lab on a Chip vol. 10 (8), (2010) a microfluidic chip to determine the concentration of sperm in the semen. For the method, a microchannel with a pair of planar electrodes that allows the detection of sperm passing through the electrodes, using electrical impedance measurements. It is also described that cells that are not sperm in the semen also cause a change in impedance when passing through the electrodes, interfering with the sperm count. The change in electrical impedance is related to the size of the cells that pass through the electrodes, allowing the differentiation between sperm and HL-60 cells suspended in the washing medium or polystyrene beads. SUMMARY OF THE INVENTION
[009] Artificial insemination (AI) is a well-established technique in the animal industry for herd production. The selection of sperm samples for AI is based on sperm concentration, motility and cell morphology. All factors had an impact on the success rate of fertilization and the abundance of offspring. Therefore, AI centralizes satisfaction to high standards to provide high quality sperm samples to ensure high probability of fertilization after AI. Some examples of criteria for sperm sample rejection are low sperm cell motility (less than 60% progressive motility or 70% motility (both for pigs and livestock), in the fresh sample), in low general concentration, and a low overall concentration. high number of morphologically abnormal sperm cells (> 15-20%). A sperm defect that occurs frequently is the presence of a cytoplasmic drop in the flagellum of the sperm. This drop is part of the cytoplasm of spermatids, which was not removed from the flagellum at the end of spermiogenesis. Cytoplasmic drops are commonly found in one of two positions. Near the head of the sperm cell, proximal cytoplasmic drops can be found, while so-called distal drops may be present in the tail, far from the head. Although the effect of residual cytoplasm retention on human infertility is a clinically controversial issue, many sources show the contribution of gout content, especially distal gout content, to subfertility in domestic species. Therefore, sperm samples (cattle and pig) containing more than 20% of cells with cytoplasmic drops are taken (in general) from the AI. In the selection process, a high number of healthy, morphologically normal sperm cells are discarded. Unfortunately, routine sperm refinement techniques, such as sperm density centrifugation and sperm fluctuation, are not suitable for the recovery of these sperm cells for AI purposes.
[010] A potential approach to obtaining these healthy and morphologically normal sperm cells from discarded samples is the use of microfluidic technology. Microfluidic systems have been used for the manipulation, analysis and enrichment of viable motile sperm cells.
[011] However, known systems are not intended to be able to perform sperm analysis and selection based on cell morphology (at the single cell level). The classification of morphologically normal and morphologically abnormal, as sperm cells containing cytoplasmic gout, is not a simple process, as both species are very similar. A plausible criterion to differentiate these species is the total cell mass, since abnormalities can affect, and, especially, cytoplasmic gout content will affect this property.
[012] With this, it is an aspect of the invention to provide an alternative system for the classification of biological cells and, especially, sperm cells, which, still preferably, at least partially avoids one or more of the disadvantages described above. At least part of the system can be comprised on a chip. It is an additional aspect of the invention to provide a method for classifying sperm cells, especially to perform sperm analysis and selection based on a cell characteristic, especially cell morphology (at the single cell level), preferably with avoid at least partially one or more of the disadvantages described above.
[013] With this, the invention provides, in a first aspect, a system, especially for classifying (a) sperm cell (s) in a fluid, the system comprising (i) a fluid flow channel for carrying said fluid, the fluid flow channel comprising an inlet, an analysis zone configured downstream of said inlet and comprising a first pair of electrodes comprising a first intra-electrode distance, a classification zone configured downstream of said zone of analysis, and (at least two) outputs configured downstream of said classification zone, (ii) an electrical source configured to provide an electrical signal to the first pair of electrodes, (iii) a measuring device operatively coupled to the first electrode pair and configured to measure a first impedance as a function of fluid time between the first pair of electrodes, and to provide time-dependent impedance data; (iv) a classification device configured to classify sperm cells by directing a sperm cell in the classification zone to one of the outputs based on a comparison in a time-dependent impedance data comparison stage with predefined reference data.
[014] In particular, the system comprises a system for classification and differentiation of sperm cells based on sperm cell characteristics, for example, sperm cell morphology, DNA integrity (sperm cell), abnormalities within the sperm, such as vacuoles, deficiency of acrosome etc. In particular, the system comprises a system for performing sperm analysis and selection based on (a) sperm cell sperm cell characteristic (s) (see additionally below).
[015] In the realizations, the system can allow the differentiation of sperm cells having different sperm cell morphologies from one another and, especially, successive classification of the differentiated sperm cells. In particular, the system comprises a system for performing sperm analysis and selection based on sperm cell morphology of sperm cells (at the single signal level).
[016] Especially, here, the term "classification", as in "classification of a sperm cell", can refer to the differentiation of sperm cells and, especially, physical classification in a successive way, that is, separating one sperm cell from another sperm cell (especially, based on a characteristic of the sperm cell). In particular, the term "classification of a sperm cell" can refer to the performance of sperm analysis and selection based on a characteristic of the sperm cell, especially sperm cell morphology. In particular, the term "classification of a sperm cell" can refer to the classification between a first sperm cell and an additional sperm cell, based on a characteristic, especially based on a presence of the characteristic or a value of the characteristic.
[017] Especially, this system allows sperm analysis and selection based on sperm cell characteristics (abnormal), such as cell morphology (abnormal), especially at the single cell level. In particular, the system can be applied to detect a sperm cell comprising a specific abnormality or characteristic, such as an abnormal sperm morphology (cell), in the sperm cell analysis and separation zone comprising the abnormalities or specific characteristic of a fluid comprising sperm cells. , especially, a sperm cell in a classification zone configured downstream of the analysis zone. The success of differentiation, especially in relation to cell morphology, with this system can be more than 65%. Thus, this system can be used for fast, high-quality selection, leading to substantially less disposal of healthy, morphologically normal sperm cells that would otherwise be removed for AI. An advantage of the microfluidic system is that it can be scaled easily, as by parallel processing on the same chip. Thus, for classification between a normal (morphological) sperm cell (especially, a sperm cell comprising a normal sperm morphology (cell)) and an abnormal sperm cell or a sperm cell comprising a (determined) characteristic, the system can especially understand two exits, such as a first exit and a second exit. Especially, for classification between a normal sperm cell, an abnormal sperm cell and, for example, other particulate material, the system can comprise at least three outlets. A particulate material that can be comprised in sperm is, for example, waste. A sperm cell, in particular, is (also) a particulate material. Thus, a particulate material can comprise a sperm cell. A particulate material may comprise especially other particulate material, especially, another particulate material may not comprise a sperm cell. Then, the fluid flow channel may comprise two or more outlets for classification of sperm and / or other particulate material. In particular, the terms "normal" and "abnormal", as in "a normal sperm cell" and "an abnormal sperm cell", can refer to a characteristic (see further below) of the sperm cells (such as the absence or presence of a cytoplasmic gout) Especially, an abnormal sperm cell comprises an anomaly.
[018] The fluid may, in embodiments, comprise, for example, undiluted semen. Alternatively or additionally, the fluid can still comprise other liquids. The fluid may, for example, comprise diluted semen. As a diluent, for example, water, it can be applied, optionally in combination with one or more salts and / or dissolved sugars, as in the case of a Beltsville Thawing Solution. The fluid can comprise diluted semen, in a range of, for example, 10-10,000 times dilution. In embodiments, the sperm concentration in the fluid is selected from the range of 104-5,108 ml-1 cells. In particular, the fluid comprises a liquid. Especially, the fluid allows the movement of the sperm cell (in the fluid) through the system, especially through the fluid flow channel. The fluid (comprising a sperm cell) can be provided at the inlet of the fluid flow channel, wherein the fluid can additionally flow through the fluid flow channel towards an outlet disposed downstream (during normal operation) of the inlet. The inlet is specially configured to allow a fluid to enter the fluid flow channel. The inlet may be fluidly connected to another fluid channel. The inlet may also be in fluid connection with a container (reservoir) comprising the fluid. In particular, the inlet is in fluid connection with the means for supplying the fluid (comprising a sperm cell), especially to provide a fluid flow in the fluid flow channel. The entry can comprise an entry. The entry can also comprise more than one entry. Then, the system can also include a pump configured to provide a flow of fluid through the fluid flow channel.
[019] The analysis zone is specially configured to allow fluid (comprising a sperm cell) to flow through the analysis zone (see also below) and to analyze (detect or measure) a characteristic of the fluid comprising the sperm cell, or the sperm cell per se, (flowing) in the analysis zone. The system can be provided with a sensor to detect the characteristics of the fluid (flowing) in the analysis zone. In particular, the sensor comprises the measuring device described herein configured to measure a first impedance (and a second impedance). The system, especially the analysis zone, can comprise a (first) pair of electrodes to analyze a characteristic, such as the electrical impedance, of the fluid (flowing) in the analysis zone. In addition or alternatively, the system may comprise an optical sensor and / or an acoustic sensor to detect or analyze the characteristic of the fluid (flowing) in the analysis zone. Based on the analysis, the sperm cell (and part of the fluid) can be directed to a (specific) outlet in the classification zone.
[020] The classification zone is specially configured to allow the targeting of a sperm cell and / or fluid comprising the sperm cell to an outlet, especially to separate the sperm cell from other sperm cells. In particular, the system comprises a sorting device configured to direct the sperm cell in the sorting zone to one of the exits. Consequently, the targeting of a sperm cell to one of the outlets may comprise the targeting of the sperm cell in the fluid flow to one of the outlets. For example, the targeting of the sperm cell by dielectrophoretic forces or other means, especially to direct material (a particulate) in a fluid. Alternatively or additionally, directing a sperm cell to one of the outlets may comprise directing the fluid comprising the sperm cell to one of the outlets. The targeting of the fluid (comprising the sperm cell) may, for example, comprise targeting the fluid flow by means of additional valves or fluid flows or other (hydrodynamic) means especially to direct the fluid flow (comprising the sperm cell). Then, the classification device may comprise a device for providing dielectrophoretic forces (to the sperm cell) or other means for directing particulate material, such as a sperm cell, in a fluid in the classification zone. In particular, the system may comprise electrodes to provide dielectrophoretic forces. Alternatively or additionally, the sorting device may comprise a device for directing the fluid comprising the sperm cell to one of the outlets, for example, by means of valves or other means, in particular, to direct a fluid flow. In particular, the classification device may comprise a valve.
[021] A (first) impedance, as described here especially refers to an (first) electrical impedance. Especially, the impedance, as described here, can refer to the absolute electrical impedance. The (electrical) impedance is especially the fluid (volume) response between the electrodes of the electrode pair that can be measured when a voltage (potential) difference (AC or DC, especially AC) is introduced by the (first) pair electrodes to provide a current (flowing through the fluid, especially between the electrodes of the pair of electrodes) (see below). In particular, an electrical signal is provided to the first pair of electrodes (and, optionally, a second pair of electrodes, see below). Therefore, the electrical impedance is especially a response to an electrical signal provided to the electrodes of the pair of electrodes. The electrical impedance is especially affected by the dielectric characteristics of the (volume of) fluid between the electrodes. Thus, if a fluid flow between (the electrodes of) the pair of electrodes, the characteristics, such as the dielectric characteristics, of the volume that is measured between the electrodes can change over time and, especially, the measured impedance can change over time. Consequently, "measurement of a (first) impedance as a function of fluid time between the (first) pair of electrodes" comprises the measurement of the electrical impedance (or signal) value (response of a potential difference by the electrodes) of the fluid (including any optional sperm cell and other optional material) between the electrodes of the electrode pair for a period of time (and determining the impedance (signal) values as a function of time in that period of time). Impedance (electrical) (value / signal) is affected by the dielectric characteristics of the fluid (including a possible sperm cell or other particulate material). Thus, when an abnormal sperm cell or a sperm cell comprising a (specific) characteristic, especially a sperm cell presenting a (abnormal) characteristic of the cells, affecting the dielectric characteristics of the sperm cell, it flows between the pair of electrodes and the impedance (electrical ) is measured over time, the measured signal (impedance) may differ substantially from an observed form of a normal sperm cell or a sperm cell not comprising the (specific) characteristics and the abnormal sperm cell / sperm cell comprising the (specific characteristics) ) can be identified.
[022] In particular, identification can be done at a comparison stage (or identification stage). Cell characteristics that affect the dielectric properties of a sperm cell, for example, include changes in the charge on the sperm membrane, distribution of charge across the sperm membrane and abnormal morphology. In particular, abnormal morphology, such as size variations, vacuoles in the head, or acrosome deficiency, and the presence of a cytoplasmic drop can have a substantial effect on the measured impedance signal when the abnormal sperm cell passes between the electrodes.
[023] The system and method described here can be applied to detect and classify a sperm cell comprising an abnormal or specific characteristic affecting the dielectric characteristics of the sperm cell. In particular, the system and method can be applied to detect and classify a sperm cell comprising a morphological abnormality, such as a sperm cell comprising a cytoplasmic drop. The system can also be configured to (and the method as described here can also comprise) select the presence of a (other) specific characteristic having an effect on the dielectric characteristics of a sperm cell, for example, (the presence of) abnormal dimensions of a sperm cell, abnormal vacuoles in the sperm cell, abnormalities in the acrosome, abnormalities in the sperm membrane load, or (the presence of) other morphological abnormalities. So, especially the system of the invention can be configured, and the method of the invention can be used, for classification of a sperm cell based on one or more characteristics. In particular, the characteristic can be selected from the group consisting of a dimension of the sperm cell, the presence of (a certain) vacuole in the sperm cell, an acrosome (deficiency) (in the sperm cell), a charge from a sperm cell membrane (a sperm membrane), a charge distribution across the sperm membrane, a morphology (a morphological characteristic) (of the sperm cell), the presence of a cytoplasmic drop, and the integrity of DNA).
[024] Here below, the system is described in more detail. The system (and method) can also be used to (only) measure or analyze sperm. In particular, to analyze the sperm, the classification device may not be applied to classify the sperm (separating a sperm cell from another sperm cell) in the classification zone. Especially, for this application, there is no need for a classification zone (and classification device). The system (and method) is explained especially in more detail for identification and classification of a morphologically abnormal sperm cell, especially a sperm cell comprising a morphology that differs from the morphology of other sperm cells.
[025] When an abnormal sperm cell flows between the pair of electrodes and the impedance is measured over time, the measured signal may differ substantially from that observed from a normal sperm cell. Thus, an abnormal sperm cell (and also the normal sperm cell) can be identified and differentiated (especially, in the comparison stage). In particular, the system (and method) described here is (configured) for identifying and differentiating a sperm cell based on a sperm cell characteristic. The identified (abnormal) sperm cell can be directed to one of the outputs by the classification device, while a normal sperm cell (or sperm cell not comprising specific characteristics) can be directed to (one of the other) output (s). An outlet may comprise an opening for exiting the fluid flow channel. An outlet can be fluidly connected to one or more additional fluid flow channels, for example, to direct the selected sperm cell to an additional processing or storage stage. Alternatively or additionally, one or more of the outlets may be in fluid connection with a container, especially to contain (a fluid comprising) the classified sperm cells. In one embodiment, one of the outlets is configured as a continuation of the fluid flow channel, while the other outlet (s) is (are) configured (s) as a side or outlet of the flow stream channel. main fluid. Especially, in this embodiment, a standard fluid flow can be provided from the inlet to the outlet, configured as a continuation of the fluid flow channel and an identified sperm cell (and / or any other identified material, see below) can be removed from the main fluid flow by directing it (the identified sperm cell and / or other material) to the outlet (s) configured as an outlet. In other words, a channel axis upstream of this "continuation" output and a channel axis downstream of it can be configured substantially in parallel and substantially without a mutual distance (especially, substantially mutual in line with one another).
[026] Especially, in an embodiment comprising at least three exits, the system can still be configured for an additional classification, for example, classification between a normal sperm cell, a sperm cell comprising a distal cytoplasmic drop, and a sperm cell comprising a proximal cytoplasmic gout. The sorting device can also be configured to direct the normal sperm cell, for example, to a first outlet, and abnormal sperm cells (comprising, for example, a distal cytoplasmic drop and a proximal cytoplasmic drop) can be directed to one of the respective second and third outputs and, optionally, additional outputs (based on a comparison in a stage of comparison of the time-dependent impedance data with predefined reference data). The outlets can all be configured in substantially the same location in relation to the fluid flow channel axis, for example, as in the case of a bifurcation or a trifurcation. In particular, the fluid flow channel axis comprises the longitudinal axis of the fluid flow channel. In particular, the fluid coming out of the different outlets can be directed in different directions of flow. Especially, in this realization, a sorting device can be configured to direct the sperm cell to any of the outputs. Alternatively, more than one sorting device can be configured to direct the sperm cell to a specific outlet.
[027] In an additional or alternative way, the exits can also be arranged in series. For example, for the classification of the three types of sperm cells mentioned above, it may be advantageous to first classify between normal and abnormal sperm cells and direct all normal sperm cells to a first outlet and all abnormal sperm cells to a second outlet. The second outlet can be configured downstream from the first outlet. Downstream of the second outlet, but on the same channel as the first outlet, one or more additional outlets can be configured. Alternatively or additionally, this second outlet may comprise one or more additional outlets, such as a third or additional outlet, configured downstream of the second outlet in another channel, different from the channel comprising the first and second outlets. Different embodiments can be used to successively direct normal sperm cells to a first outlet and abnormal ones, for example, to a second outlet, for example, with sperm cells comprising, for example, a distal cytoplasmic drop to an outlet additional (specific). In another embodiment, the sorting device can be configured to direct a normal sperm cell and an abnormal sperm cell, respectively, to a first and a second outlet, while the material (non-sperm cell comprising) additional (particulate) material (such as waste) cells) comprised in the fluid can be directed to yet another (additional) outlet.
[028] Consequently, in an additional embodiment, the system comprises (a first exit, a second exit and) an additional exit, and the classification device is further configured to classify additional particulate material by directing the additional particulate material in the zone. classification to one of the (first, second or additional) outputs, especially based on a comparison in a stage of comparison of the time-dependent impedance data with predefined reference data. In particular, reference data may comprise reference data for additional particulate material. This system can be especially relevant when the fluid can additionally comprise an additional particulate material (in addition to the sperm cells).
[029] In the realizations, the classification device is configured to classify sperm cells between normal sperm cells (morphological) and abnormal sperm cells (morphological), by directing the sperm cell in the classification zone to one of the outputs based on a comparison in a stage of comparison of time-dependent impedance data with predefined reference data, in which an abnormal sperm cell is directed to one of the outputs by the classification device and a normal sperm cell is directed to another output.
[030] The electrical source can supply an alternating current to the electrodes. The electrical source can also provide a direct current to the electrodes. The electronic source can also provide the electrical signal comprising waves, such as a sine wave, a blocking wave, or a triangular or toothed wave. In one embodiment, the electrical source is comprised of a classified electrical device. In yet another embodiment, the electrical source may also be included in the measurement device (see below). In a specific embodiment, an impedance spectroscope comprises the measuring device as well as the electrical source. However, in yet another embodiment, an electric wave generator comprises the electrical source, and the measuring device comprises an impedance spectroscope.
[031] The time-dependent impedance data provided can comprise different representations. Time-dependent impedance data can, for example, in one embodiment, comprise a series of data (such as a table) comprising the raw measurement data, that is, the (measurement) time and the respective measured impedance signal, especially, the respective imaginary and real part of the measured signal. In another embodiment, the time-dependent impedance data may comprise a single value, such as the maximum measured impedance signal (when measuring the impedance of a fluid comprising a sperm cell) or, for example, an impedance increase level ( for a specific measurement period). In a further embodiment, the time-dependent impedance data may comprise a series of data comprising the minimum and maximum measured impedance values and the respective measurement moments. In yet another embodiment, the time-dependent impedance data may comprise a graphical representation of the measured impedance versus the measurement time, especially configured as a measurement curve. Also, in an additional realization, the measured impedance values are transformed into absolute impedance data and the time-dependent impedance data can comprise the absolute impedance values (and the respective measurement time). In particular, the absolute value of the measured impedance data can be used. In particular, in realizations, the time-dependent impedance data comprises the absolute values of the measured impedance signal (as a function of the measurement time). In yet another realization, the time-dependent impedance data can comprise the real and imaginary parts of the measured impedance values. Alternatively or additionally, the measured impedance signal can be corrected in relation to the derivation and / or deviation in the measured data; time-dependent impedance data can (also) comprise corrected data. Also, in an additional realization, the time-dependent impedance data comprises at least two of the representations given above (realizations). Then, the measuring device can be specially configured to provide different types of time-dependent impedance data, such as the data in the above achievements. Time-dependent impedance data can therefore include originally measured data as well as processed data. In addition, time-dependent impedance data can include a plurality of data or, optionally, up to a single value.
[032] When a normal sperm cell (or other particulate material) flows between a pair of electrodes and the fluid impedance (comprising the sperm cell) between the electrodes is measured over time, time-dependent impedance data can be compared to predefined reference data in different ways. In embodiments, the comparison may comprise different types of analysis of mathematical and / or statistical data known in the art. Time-dependent impedance data can comprise (first) measured (gross) impedance data (gross) over time and can, for example, be analyzed and compared directly to predefined reference data. Time-dependent impedance data can also, at the comparison stage, be transformed and / or visualized by techniques known in the art and compared to predefined reference data. In one embodiment, the data can be stored in a (temporary) memory and only the highest value is used to compare with predefined reference data. In another embodiment, the minimum and maximum time-dependent impedance values are compared to the predefined reference data, especially, the difference between the maximum and minimum impedance value can be calculated (in the comparison stage) and compared to the values comprised by the data predefined reference points. In addition, in a further realization, the time-dependent impedance data comprises a (corrected) measurement curve (data) and the predefined reference data may comprise a reference impedance curve of a normal sperm cell and / or a reference (data) of an abnormal sperm cell, and the measurement impedance curve can be compared to the reference impedance curve (s) or the data of the reference impedance curve (s). Alternatively or additionally, the measurement curve can also be provided to the model with the best fit of the measured data and compared to a mathematical model of a reference impedance curve of a normal (morphologically) sperm cell and / or a mathematical model of the reference curve of a morphologically abnormal sperm cell or different types of morphologically abnormal sperm cells or even other particulate material. A measurement curve can be represented by data and a graphical representation of the data. Especially, in realizations, a measurement curve can comprise data as well as a graphical representation, especially, where the data can be used for comparison purposes and, especially, the graphical representation for illustrative reasons. However, in other realizations, in an alternative or additional way, the graphical representation can be used for comparison purposes (only).
[033] The invention also includes embodiments using alternative comparison techniques. If the impedance is measured over time when a sperm cell passes between the electrodes, at the measured signal (impedance) (over time), an increase and a decrease in the impedance caused by the head of the sperm cell can be observed. The increase and decrease in impedance can be (graphically) represented by a peak. For a normal (morphologically) sperm cell, a subsequent tail of the sperm cell passing between the electrodes can only have an effect on the impedance signal measured between the electrodes to a very limited degree. Thus, a morphologically normal sperm cell can only substantially show a gradual increase followed by a gradual reduction in the measured signal (impedance) (over time), in which the reduction may show some enlargement. The peak can be substantially symmetrical, especially when using a homogeneous electric field between the electrodes. Here, symmetrical means that the peak or curve has symmetry on an axis, that is, the leading edge of the peak has approximately the same shape (but mirrored) as the trailing edge of this peak. In addition, several normal (morphologically) sperm cells can present an equal substantial measured signal (impedance). In addition, (measurements of) different normal sperm cells may have substantially the same shape as the measurement curve (over time), especially when the sperm cells flow substantially through the same location between the electrodes. In particular, the term “measurement curve” refers to a measured impedance signal as a function of time.
[034] When an abnormal sperm cell (morphologically) flows between the pair of electrodes and the impedance is measured over time, the measured signal (impedance) can differ substantially from that observed in the normal sperm cell. In particular, an abnormality in the form of the presence of a cytoplasmic droplet, for example, can show substantially the same peak on the measurement curve, as shown by a normal sperm cell. However, in addition to the substantial symmetrical peak, an additional (small) peak may be present in the measurement curve caused by the (extra) impedance induced by the cytoplasmic drop. Therefore, the cytoplasmic gout can be represented by a (second) small identifiable peak. The cytoplasmic gout can also be identified by an asymmetry or extra border on the measurement curve. In particular, the second peak can be superimposed on the first peak and can be identified by an edge (peak) on the measurement curve. Then, the system and method described here allow to identify the presence of the cytoplasmic gout and additionally direct the abnormal sperm cell to another outlet of the fluid flow channel (different from which the normal sperm cell can be directed). In particular, an abnormal sperm cell comprises a (pre) determined characteristic, especially a cytoplasmic gout. In particular, the system and method, as described here, can identify the presence of the cytoplasmic gout based on an asymmetric measurement curve to further target the abnormal sperm cell to one of the outlets. In particular, the system and method, as described herein, can identify a normal sperm cell based on a substantially symmetrical curve to further route the normal sperm cell to one of the outlets, especially while directing other particulate material to the other outlet.
[035] To facilitate the positioning of the sperm cells (between the electrodes), an optional focusing zone can be configured upstream of the analysis zone. In particular, the focus zone is configured to direct a sperm cell to a specific location in the fluid flow, especially towards a central axis of the fluid flow channel, especially towards the axis of the fluid flow channel ( location of the focus zone). The targeting of sperm cells can be provided by special adaptations in the flow channel, such as small restrictions or narrowing of the flow channel. Focusing can also be provided by the application of ultrasound. However, it has been found that sperm cells can be advantageously positioned without losing viability by subjecting the sperm cell to a non-uniform electric field. The invention thus also provides that the positioning of a sperm cell within the flow channel can be controlled by dielectrophoretic forces (in the focus zone).
[036] Also, in the classification zone, a sperm cell can be advantageously directed to a specific outlet providing dielectrophoretic forces to the sperm cell.
[037] Consequently, dielectrophoretic forces can be provided in the focusing zone and / or in the classification zone to target a sperm cell. Dielectrophoretic forces can be provided especially in the classification zone and even more especially in the focusing zone and in the classification zone.
[038] Then, in one embodiment, the system, especially the sorting device, comprises a first electromagnetic device to provide an electric field to the sorting zone, and the first electromagnetic device is configured to direct the sperm cell by a dielectrophoretic force (dielectrophoresis) (in the classification zone). In this way, the classification of sperm cells can be performed.
[039] In an additional embodiment, the system comprises a second electromagnetic device to provide an electric field to the focus zone. In particular, the second electromagnetic device is configured to direct the sperm cell by dielectrophoretic force, especially to the axis of the fluid flow channel. In this way, sperm cells can be forced to flow, for example, substantially in the middle of the fluid flow channel axis.
[040] For example, the dielectrophoretic force can be provided by applying an electric field in the MHz range using the microelectrodes integrated in the chip. In particular, cell focusing and classification can be accomplished by applying a sinusoidal excitation of 10 MHz, 6VPP by the first and / or second electromagnetic device and the first and / or second electromagnetic devices are configured to provide these excitations. In particular, a dielectrophoretic force can be provided by at least two electrodes. In particular, the electrodes may be in physical contact with the fluid (in the fluid flow channel). In one embodiment, the dielectric strength is provided by two electrodes, especially when applying an AC or DC electric field, especially an AC electric field, between the two electrodes. In particular, a sperm cell or other particulate material can be directed in the direction (or opposite to the direction) of the field lines of the electric field. In an additional embodiment, the dielectrophoretic force is provided by four electrodes, especially a first set of two electrodes and a second set of two electrodes, especially, in which a first electric field (AC or DC) is applied between the first set of two electrodes and a second electric field (AC or DC) is applied between the second set of two electrodes. In particular, by arranging the first set of electrodes upstream of the second set of electrodes, a dielectric force can be provided to target a sperm cell. In particular, the two electrodes of the first set (of electrodes) can be configured at 0 ° and 180 ° respectively in relation to the fluid flow axis. In particular, the two electrodes of the second set (of electrodes) can be configured respectively at 90 ° and 270 ° with respect to the fluid flow channel axis. The two sets of electrodes can be configured at least in a plane perpendicular to the axis of the fluid flow channel. In particular, the two sets of electrodes can be configured in two planes perpendicular to the axis of the fluid flow channel, especially each set in a plane perpendicular to the fluid flow channel. Especially in this way, a sperm cell can be directed in a plane perpendicular to the axis of the fluid flow channel. In particular, a sperm cell can be directed to the axis of the fluid flow channel. In an additional embodiment, focusing is provided by ultrasound.
[041] A normal (morphologically) sperm cell (of a bull and / or a boar) can have a head size of 8-9 μm in a first direction parallel to a longitudinal axis of the head, and 4-5 μm in a second direction perpendicular to the longitudinal axis, and less than 1 μm in a third direction perpendicular to the first and second directions, and a tail of 40 -45 μm. A sperm cell comprising a cytoplasmic droplet can also be substantially the same size as the morphologically normal sperm cell, with the exception that it comprises a droplet, which is normally positioned somewhere in the middle of the sperm cell's tail (a distal cytoplasmic droplet) or behind the head (a proximal cytoplasmic drop). The head of a sperm cell is not substantially round, but it can be relatively flat, especially in the third direction. With this, it can also be advantageous to have a restriction in the fluid flow channel configured to guide the sperm cell to be analyzed with the head in a specific direction. In particular, a constraint can be configured to rotate the sperm cell around its longitudinal axis. In particular, an orientation zone, comprising this restriction, can be provided in the fluid flow channel downstream of the focusing zone and upstream of the analysis zone. Thus, in one embodiment, the system still comprises an orientation zone configured downstream of the entrance and (if present) of the optional focusing zone and upstream of the analysis zone, in which the orientation zone comprises at least one restriction (element ) in the fluid flow channel to guide the sperm cell. The orientation may comprise rotation of the sperm cell around its longitudinal axis. In an additional or alternative way, the orientation may comprise alignment (from the longitudinal axis of) the sperm cell to the fluid flow channel axis at the location of the electrode pair, especially where the head of the sperm cell is arranged further downstream from the than the tail. Then, the orientation may comprise the alignment of a sperm cell to the fluid flow channel axis, in which the head of the sperm cell is arranged more downstream than the tail (of the sperm cell) and, especially, by rotating the sperm cell around its axis to provide a substantially constant angle between the third direction of the sperm cell head and the electromagnetic field lines (between the pair of electrodes). It was realized that the orientation can also be provided by dielectrophoretic forces. With this, dielectrophoretic forces can be used to direct a sperm cell in a specific direction. Alternatively or additionally, dielectrophoretic forces can be applied to direct the position of a sperm cell to a specific location. In particular, a focusing zone comprising an electromagnetic device configured to target a sperm cell can also provide orientation of the sperm cell. Then, the functionality of the orientation zone (optional) can also be understood in the focus zone (optional). Especially in one embodiment, the second electromagnetic device (for directing a sperm cell in the focus zone) is additionally configured to orient the sperm cell (in the focus zone).
[042] The measured impedance signal can be sensitive to small disturbances (noise), present internally in the system as well as disturbances (noise) present external to the system. For example, small fluctuations in the conductivity of the fluid, in the electrical signal provided to the electrodes, or fluctuations in any electromagnetic radiation outside the system, can all have an effect on the measured impedance signal. So, it may be advantageous to provide a second pair, or even additional pairs, such as a third, fourth, fifth or even tenth pair of electrodes (downstream of the first pair of electrodes) in the analysis zone and measure the impedance at the locations successive tests in the analysis area. It can be advantageous if a pair of electrodes comprises a main electrode to connect to the electrical source and a measurement electrode to connect to the measuring device. However, the phrase “pair of electrodes” (or “pair”) does not refer only to “two” electrodes. A pair of electrodes can also refer to a pair of electrodes comprising a main electrode and two or more measuring electrodes. Likewise, two pairs of electrodes can comprise only one main (mutual) electrode and two measuring electrodes. A pair of electrodes can also refer to one or more primary electrodes and one or more measurement electrodes. In particular, a main electrode can be comprised of one or more pairs of electrodes. In particular, using two (or more) pairs of electrodes, time-dependent impedance data (for a sperm cell) can be provided based on the impedance signals measured between the first pair of electrodes and the impedance signal measured between the second electrode pair (and, if present, also between additional electrode pairs). If two (or more) pairs of electrodes are used in the system, a sperm cell can be detected multiple times and extra information about the sperm cell can be generated, which can be used to reduce the noise effect. The impedance signals measured from the two or more pairs of electrodes can, for example, be weighted to provide time-dependent impedance data (using different time stamps to correct the time required to flow from the first pair of electrodes to the successive pair (s) of electrodes) to remove part of the noise and / or to improve a possible baseline correction (deviation). However, it was surprisingly found that by using two pairs of electrodes and making differential measurements, the effect of systemic errors can be substantially reduced compared to classifying the impedance records. By subtracting (differentially) the signals from the two pairs of electrodes (in which data points measured at the same time in the first pair and in the second pair of electrodes are subtracted from each other), a systemic error that is present within the spectroscopy of impedance can be solved. In particular, by subtracting (differentially) the signals, impedance data dependent on the time of the differential signal can be provided. Using a differential signal, a pass-through sperm cell can be represented on a (time-dependent impedance data configured as) measurement curve by a positive peak (comprising positive differential impedance values) followed by a negative peak (comprising impedance values) differential differentials). The shape of the measurement curve (or any other type of time-dependent impedance data based on a difference between the measured data of the first and second electrode pairs) can then contain information about the presence of an abnormality, especially , a sperm cell comprising an abnormal morphology, such as, in particular, a cytoplasmic gout. A normal (morphologically) sperm cell may have a positive and a negative peak caused by the head of the sperm cell on the measurement curve (the differential signal). In particular, the negative peak may show peak widening caused by the tail of the sperm cell (for a sperm cell that travels the head, first, between the electrodes). However, a sperm cell comprising a morphological abnormality, such as a cytoplasmic drop, may have an additional border (or additional peak) between the negative peak and the widening of the negative peak of the differential signal (impedance). Especially, the subtraction in a differential way comprises the subtraction of a (second) data point measured at a moment in the second pair of electrodes from a (first) data point measured at said moment in the first pair of electrodes (or vice versa) . In fact, this can be done for a plurality of second data points and first data points.
[043] Then, in a specific embodiment, the invention still provides the system, in which the analysis zone still comprises a second pair of electrodes comprising a second intra-electrode distance and configured at an inter-electrode distance of the first pair of electrodes , and where (i) the electrical source is configured to still provide an electrical signal to the second pair of electrodes; and (ii) the measuring device is also functionally coupled to the second pair of electrodes and configured to measure a second impedance as a function of fluid time between the second pair of electrodes and configured to provide the impedance data dependent on the time based on the first impedance and the second impedance. The inter-electrode distance is especially defined as the shortest distance between the first pair of electrodes and the second pair of electrodes.
[044] The first pair of electrodes is specially configured to be two opposite sides of the flow channel, with the flow channel between them. Likewise, the second pair of electrodes is specially configured to be on two opposite sides of the flow channel, with the flow channel between them. In particular, the distance between an electrode in a pair of electrodes and the fluid flow channel axis is equal to the distance between another electrode in the electrode pair and the fluid flow channel axis. The first pair of electrodes and the second (optional) pair of electrodes are specially configured to be free of physical contact with a fluid flowing between the electrodes. The electrodes of the electrode pairs comprise electrically conductive material, such as a metal or other conductive material. In particular, the electrodes may comprise one or more metals selected from the group consisting of iron, copper, aluminum, gold, silver, nickel, platinum, titanium, tantalum, tin, and alloys thereof. In one embodiment, the electrodes comprise platinum and / or titanium. In an additional embodiment, the electrodes (also) comprise tantalum and / or titanium. Alternatively or additionally, the electrodes may (also) comprise graphite.
[045] Especially, this realization can be advantageously combined with an realization in which the data related to time-dependent impedance are based on a difference between an absolute value of the first impedance at a moment and an absolute value of the second impedance at that moment. However, in another embodiment, the time-dependent impedance may be based on the average values of the signals (impedance) measured from the first and second pairs of electrodes.
[046] Time-dependent impedance data based on the two electrode pairs can comprise substantially the same data as that provided with only one pair of electrodes. In addition, the time-dependent impedance data provided in an embodiment comprising two pairs of electrodes can comprise data based on the first pair of electrodes, data based on the second pair of electrodes, and data processed based on the measurement data of the first and the second pair of electrodes.
[047] Also, the reference data predefined in an embodiment comprising two pairs of electrodes can comprise the same reference data based on only one pair of electrodes, especially, the predefined impedance data based on the two pairs of electrodes can comprise reference data in relation to the first pair of electrodes, reference data in relation to the second pair of electrodes, and reference data in relation to the processed data of the first and second pair of electrodes. So, when predefining the reference data based on the representation of the time-dependent impedance data, the (method for) classification of sperm cells, in one embodiment, comprising one pair of electrodes and one embodiment comprising two pairs of electrodes, is substantially the same. In particular, the reference data may contain information (permission) to classify between sperm cells based on a characteristic, especially, to classify between morphologically normal sperm cells and sperm cells comprising a morphological abnormality, such as a cytoplasmic drop. In particular, reference data can also contain information to classify between a sperm cell and one or more other particulate materials. Then, in one embodiment, the reference data contains information about the presence and / or absence of a morphological abnormality. In an additional embodiment, the reference data contains information about the presence and / or absence of a cytoplasmic gout. In an additional embodiment, the reference data contains information about a (additional) sperm cell characteristic. Also, in an additional realization, the reference data contains information about a particulate material, especially waste. In addition, reference data may include information about a deviation from reference data within which a species to be classified belongs to a specific class (such as normal sperm or abnormal sperm) or outside which a species to be classified does not belong to a specific class (such as abnormal sperm or normal sperm). In particular, a characteristic of an abnormal sperm cell may differ from that of a normal sperm cell (as is known to the person skilled in the art).
[048] So, in one embodiment, the invention provides a system for classifying a sperm cell in a fluid, especially a system for performing sperm cell analysis and selection based on a sperm cell characteristic of sperm cells, especially, based on the sperm cell morphology of sperm cells, the system comprising: (i) a fluid flow channel for transporting said fluid, the fluid flow channel comprising an inlet, an analysis zone configured downstream of said inlet and comprising a first pair of electrodes comprising a first intra-electrode distance and a second pair of electrodes comprising a second intra-electrode distance and configured in an inter-electrode distance of the first pair of electrodes, a classification zone configured downstream of said analysis zone, (at least two) outputs configured downstream of said classification zone and, optionally, a zone of analysis focus set up downstream of said entrance and upstream of said analysis zone; (ii) an electrical source configured to provide an electrical signal to the first pair of electrodes and an electrical signal to the second pair of electrodes; (iii) a measuring device operatively coupled to the first pair of electrodes and the second pair of electrodes and configured to measure a first impedance as a function of fluid time between the first pair of electrodes and to measure a second impedance as a fluid time function between the second pair of electrodes, and to provide time-dependent impedance data based on the first and second impedances; (iv) a sorting device configured to classify sperm cells by directing the sperm cell in the sorting zone to one of the outputs based on a comparison in a time-dependent impedance data comparison stage with predefined reference data.
[049] The system can be applied to analyze and classify a sperm cell in a fluid. The system can be specially applied to analyze a sperm cell in a fluid. Especially for the purpose of analyzing a sperm cell in a fluid, the sperm cell may not necessarily have to be classified. Then, the invention also provides, in an additional aspect, a system for analyzing a sperm cell in a fluid, the system for analyzing a sperm cell comprising: (i) a fluid flow channel for transporting said fluid, the fluid flow comprising an inlet, an analysis zone configured downstream of said inlet and comprising a first pair of electrodes comprising a first intra-electrode distance and (optionally) a second pair of electrodes comprising a second intra-electrode distance and configured in an inter-electrode distance of the first pair of electrodes, (an output configured downstream of said analysis zone), and optionally a focusing zone configured downstream of said input and upstream of said analysis zone; (ii) an electrical source configured to provide an electrical signal to the first pair of electrodes and (optionally) an electrical signal to the second pair of electrodes; (iii) a measuring device functionally coupled to the first pair of electrodes and (optionally) to the second pair of electrodes and configured to measure a first impedance as a function of fluid time between the first pair of electrodes and (optionally) measure a second impedance as a function of fluid time between the second pair of electrodes, and providing time-dependent impedance data based on the first impedance and (optionally) the second impedance; and in which the sperm cell is analyzed based on a comparison in a stage of comparison of the time-dependent impedance data with predefined reference data. In particular, the analysis may include determining the characteristics of sperm cells, especially the amount of morphologically abnormal sperm cells in the sperm. In particular, the system (especially, the system for analyzing a sperm cell in a fluid) comprises a system for performing sperm cell analysis, especially, based on a sperm cell characteristic, especially sperm cell morphology of sperm cells.
[050] The dimensions of the systems described here can be specially configured to transport a fluid comprising a sperm cell and to position the sperm cell, especially in the center of the flow channel, especially in the axis of the fluid flow channel in the focusing zone. .
[051] The electrodes are also specially configured to not obstruct the flow of fluid in the flow channel. It may be advantageous to incorporate the electrodes in one wall of the fluid flow channel, or in two walls on opposite sides of the fluid flow channel axis. In one embodiment, the electrodes are integrated into the flow channel wall (in the analysis zone). Especially, the electrodes are micro-electrodes. In an additional embodiment, a small region of a fluid flow channel wall is notched and replaced with a metal or other electrically conductive material configured as an electrode to provide an integrated (micro) electrode (in the wall). In one embodiment, at least two small regions of the fluid flow channel wall are removed by notches and replaced with conductive material configured as electrodes to provide integrated (micro) electrodes (on the wall).
[052] Electrodes that provide a pair of electrodes can be specially configured at an intra-electrode distance allowing a sperm cell to pass. Especially, an electrode can comprise a width, a length, and a height. Here, the intra-electrode distance is the shortest distance between electrodes of a pair of electrodes. However, a large intra-electrode distance can have a negative effect on the sensitivity of the measurement. So, an intra-electrode difference, preferably, is in the variation of greater than (once) the size of the head of a sperm cell and not greater than 5 times the size of a head of a sperm cell. In particular, the intra-electrode distance between a pair of electrodes is in the range of 5 - 400 μm, especially 5 - 20 μm. Then, in one embodiment, the invention provides the system, in which the first intra-electrode distance is selected from the range of 5 - 400 μm, especially in the range of 5 - 20 μm. In an additional embodiment, the second intra-electrode distance is also selected from the range of 5 - 400 μm, especially in the range of 5 - 20 μm. Especially, in embodiments comprising a second pair of electrodes, the first intra-electrode distance and the second intra-electrode distance are selected to be substantially the same. In one embodiment, the first intra-electrode distance and the second intra-electrode distance are substantially the same, especially, the first intra-electrode distance and the second intra-electrode distance are substantially 10 μm. In another embodiment, the first intra-electrode distance and the second intra-electrode difference are substantially 20 μm. In yet another embodiment, the first intra-electrode distance and the second intra-electrode difference are not the same. In a specific embodiment, the wall of the fluid flow channel (in the analysis zone) comprises the electrodes. Especially, in this embodiment, the (first and second) intra-electrode distance can be equal to a specific dimension of the fluid flow channel, such as a height, or a width or diameter of the fluid flow channel.
[053] The fluid flow channel described herein can comprise a cross section (of the fluid flow channel and open for fluid flow) perpendicular to the fluid flow channel axis and comprises a first dimension of the fluid flow channel and a second dimension of the fluid flow channel perpendicular to the first dimension.
[054] In embodiments, the fluid flow channel may comprise a circular cross section or a substantially square cross section. Especially, the first dimension and the second dimension of the fluid flow channel can be substantially the same.
[055] The first dimension and the second dimension of the fluid flow channel may also differ from each other and the cross section may, for example, comprise a rectangular shaped cross section or even another type.
[056] In particular, the intra-electrode distance is configured to be substantially equal to or less than the first dimension of the fluid flow channel and / or the second dimension of the fluid flow channel (at the location of the electrode pair).
[057] The fluid flow channel is specially configured to transport sperm cells, see above. Therefore, the minimum dimensions (in cross section) of the fluid flow channel should allow a sperm cell to pass. Then, the first dimension of the fluid flow channel and the second dimension of the fluid flow channel are at least selected to allow a sperm cell in the fluid flow channel. The fluid flow channel may further comprise restrictions or other means for focusing or orienting a sperm cell. So, especially the first dimension of the fluid flow channel is selected from the range of 5 - 400 μm, especially, in the range of 5 -200 μm, especially 5 - 100 μm, such as 10 - 20 μm. The second dimension of the fluid flow channel is selected from the range of 5 - 400 μm, especially, in the range of 5 - 200 μm, especially 5 - 100 μm, such as 10 - 20 μm. Then, in one embodiment, the invention provides the system, in which the first dimension of the fluid flow channel is selected from the range of 5 - 400 μm and the second dimension of the fluid flow channel is selected from the range of 5 - 400 μm. In particular, the cross-sectional area (of the fluid flow channel) is at least 100 μm2, as in the range of 100 - 10,000 μm2. The different zones (analysis zone, classification zone, and optional focus zone, and orientation zone) in the flow channel can (all) comprise different dimensions from each other. Especially, however, the dimensions (of the cross section) of a first zone on the side downstream of the first zone, can be substantially equal to the dimensions (of the cross section) on the side upstream of a second zone that contacts the first zone and downstream of the first zone. The terms "first dimension" and "second dimension" especially refer to height and width, respectively. The fluid flow channel would have a square or circular cross section, so the first dimension and the second dimension would be identical.
[058] For a small distance between the electrode pairs, a significant part of a sperm cell, such as the head of the sperm cell, may be present between (or detected by) the first pair of electrodes as well as between the second pair of electrodes . The impedance measurement between the first pair of electrodes can affect the impedance measurement between the second pair of electrodes, and vice versa. Especially, the inter-electrode distance is the shortest distance between (one electrode) the first pair of electrodes and (one electrode) the second (or additional) pair of electrodes. Especially, if the first pair of electrodes and the second pair of electrodes comprise the same (mutual) electrode (opposite), the inter-electrode distance is the shortest distance between an electrode, and the mutual electrode is not the first electrode pair, and an electrode, not the mutual electrode, of the second (or additional) pair of electrodes. Large inter-electrode distances require a more extensive analysis zone and can lead to loss of information, as measurements can become more sensitive to deviation. Large inter-electrode distances may also require reduced throughput, especially when making differential measurements. In particular, it can be advantageous if the two pairs of electrodes are configured at an inter-electrode distance in which the measurement of the first impedance does not affect the measurement of the second impedance, especially where the distance between the two electrodes is minimized. Then, in one embodiment, the inter-electrode distance is selected from the range of 10 - 100 μm, especially 15 - 60 μm, such as about 20 - 40 μm.
[059] The chip can be especially a PDMS chip. Therefore, the fluid flow channel can be comprised of a chip (PDMS).
[060] In a second aspect, the invention provides a method for classifying sperm cells, especially between sperm cells comprising a (determined) characteristic and sperm cells not comprising the (determined) characteristic, especially between normal and morphological sperm cells abnormal sperm (morphological) cells, where the method for classifying sperm cells comprises: providing a fluid flow comprising a sperm cell to a fluid flow channel, where the fluid flow channel comprises a first pair of electrodes ; optionally, focusing the sperm cell on the fluid flow channel; providing an electrical signal to the first pair of electrodes and measuring a first (electrical) impedance (signal) as a function of fluid time between the first pair of electrodes to provide time-dependent impedance data; and classification of sperm cells based on the comparison of time-dependent impedance data with the predefined reference data in a comparison stage. In particular, the classification may comprise physically separating the sperm cells. In particular, the classification is based on a characteristic of a sperm cell, especially to determine a sperm cell morphology. So, the method is an ex vivo method.
[061] In the method, the (electrical) impedance (signal) is measured. In particular, time-dependent impedance data comprises the first impedance as a function of time. Consequently, sperm cells can be classified based on the comparison of time-dependent impedance data with predefined reference data, especially predefined reference data comprising characteristics of an abnormal sperm cell and characteristics of a normal sperm cell. In particular, the classification of sperm cells (between an abnormal and a normal sperm cell) can be based on comparing (the symmetry) of a measurement curve comprising time-dependent impedance data with (the symmetry of) a symmetric curve. In particular, the classification of sperm cells may comprise the classification between a sperm cell not comprising a cytoplasmic gout and a sperm cell comprising a cytoplasmic gout. Then, the invention provides a method including (a) an optional focusing stage, (b) an analysis stage, (c) a comparison stage, and (d), optionally, a classification stage, in which the Analysis of the sensor device, as described here, is applied, in particular, the sensor device comprising the measuring device configured to provide time-dependent impedance data.
[062] Especially, the method may comprise the use of the system for classifying a sperm cell, as described here. In particular, the system for classifying a sperm cell may comprise the method described herein.
[063] In one embodiment, the fluid comprises boar sperm cells. In another embodiment, the fluid comprises cattle sperm cells, especially bull sperm cells. In particular, the fluid in the method and the system for classifying sperm cells, as described herein, comprise sperm cells at a concentration of 2-103 - 2-108 cells / ml.
[064] Impedance spectroscopy is known in the art for analysis without labeling adherent cells or cells in suspension. This technique has been used extensively to investigate the dielectric properties of cells in microfluidic systems. When a sperm cell is introduced between a pair of electrodes, the capacitive and resistive properties will be altered by the cell membrane (capacity) and the cell's cytoplasm (resistance cyt), respectively. A significant effect of a double layer on the absolute impedance can be shown. Due to a small electrode surface area, impedance can decrease over a wide frequency range. However, based on the properties of the electrodes, the flow channel and the fluid (comprising a sperm cell), at a specific frequency, a resistive level can be formed. It was presented that, in the system and in the method, as described here, a measurement frequency of 1.3 MHz is an appropriate choice for analyzing sperm impedance in this configuration. In one embodiment, the method is provided, in which the impedance measurement comprises the measurement of the impedance at a frequency of 1.3 MHz. In addition, it may be advantageous to apply multiple frequencies at the same time, especially frequencies that do not interfere with each other . Then, in one embodiment, the method is provided, in which the impedance measurement comprises the measurement of the impedance at a selected frequency from the range of 10 kHz - 100 MHz. Here, the term “frequency” can also refer to a plurality of (different) frequencies.
[065] The sperm cell in the fluid flow can be directed, especially towards the center of the fluid flow channel, especially the fluid flow channel axis. In particular, the targeting (focusing) of the sperm cell in the center of the fluid flow channel can improve the reproducibility of the method. The targeting of a sperm cell may comprise the targeting of the sperm cell in the fluid flow. You can also understand the direction of fluid flow including the sperm cell. Thus, in a realization of the method, the focusing of the sperm cell comprises the provision of an additional fluid flow and a directing of liquid to the fluid flow channel. In addition, in one embodiment, the system further comprises one or more additional inlets in the focusing zone configured to provide additional flow of a targeting liquid in the fluid flow channel. The provision of an additional targeting liquid can advantageously also dilute the fluid in the fluid flow channel. Especially, in a more diluted fluid, the sperm cells can be transported further away (in a longitudinal direction in the flow channel) from each other, which can positively affect the measurement. Alternatively or additionally, the sperm cell can be targeted in the fluid, especially by applying dielectrophoretic forces to the sperm cells. Then, in an additional embodiment, a non-uniform electric field is provided to the focusing zone, and the focusing of the sperm cell comprises the provision of a non-uniform electric field to the sperm cell to direct the sperm cell in the fluid flow. In particular, a non-uniform electric field comprises a dielectrophoretic force.
[066] In particular, a non-uniform electric field can also be used to direct a sperm cell in the classification zone to one of the outlets of the fluid flow channel. Consequently, in a further realization of the method, the targeting of the sperm cell in the classification zone comprises the provision of a non-uniform electric field to the sperm cell to redirect the sperm cell in the classification zone.
[067] In an advantageous embodiment, the method may comprise measuring the (electrical) impedance (signal) at two locations in the fluid flow channel and using the signal from the two locations to classify sperm cells. Then, in a further embodiment, the invention provides the method, in which the fluid flow comprising a sperm cell is provided to the flow channel and the flow channel comprises the first pair of electrodes and a second pair of electrodes, and in which the method further comprises providing an electrical signal to the second pair of electrodes and measuring a second impedance as a function of fluid time between the second pair of electrodes; and provision of time-dependent impedance data based on the first and second impedances.
[068] When using two pairs of electrodes, it can be advantageous if the time-dependent impedance data comprises a differential signal (curve) (see also above) and to classify based on the differential signal (curve) (data). Consequently, in one embodiment, time-dependent impedance data comprises differential signal (curve) data, where differential signal (curve) data is provided by subtracting a second impedance as a function of time from the first impedance as a function of said (same) time, and the predefined reference data comprise reference data based on differential signal data (curve) from normal sperm cells and reference data based on differential signal data (curve) from abnormal sperm cells.
[069] It was surprisingly found that by systematically processing the differential signal curve data, morphologically abnormal sperm cells, especially comprising a cytoplasmic drop, can be separated from normal morphological sperm cells. It was found that, especially after processing the differential signal curve (providing a processed differential signal curve), an area below the processed differential signal curve of sperm cells comprising a cytoplasmic drop differed significantly from the area below the signal curve differential processed from normal sperm cells. As mentioned above, reference is made here to a measurement curve and an area; as will also be understood, the measurement signal (data points) can be used, processed and integrated for a specific measurement time to end with a value comparable to the area mentioned above. However, to explain the realization, a more graphic interpretation is given by using the terms curve, peak, etc. The comparison of the area under the processed differential signal (curve), as mentioned in the realization, the method comprises the following steps: - subtraction of the measured signal (absolute impedance values) (data points) of the second pair of electrodes at a given time (measurement) of the measured signal (absolute impedance values) (data points) of the first pair of electrodes at the same time (measurement), for a relevant period of time (in which the relevant period of time is selected and may be the period time in which the presence of a sperm cell is measured by the first and / or the second pair of electrodes) to provide data (impedance) dependent on the differential signal time data (impedance versus measurement time) (graphically represented by a curve of differential signal comprising a positive peak and a negative peak); - determination of B, where B is the minimum signal (impedance) value (or the peak “height” of the negative peak) of the data depending on the time of the differential signal; - determination of the measurement time (second), where the impedance value dependent on the time of the differential signal is equal to zero (graphically, the moment at which the first (positive) peak ends and the second (negative) peak begins) and the corresponding measurement time in B (the measurement time at least of the impedance value dependent on the time of the differential signal); and calculates XB, as the difference of those time values; - processing of data dependent on the differential signal time by dividing all impedance values by B, and all time values measured by XB, providing the processed differential signal data and graphically the processed differential curve; - calculation of the area under the differential signal curve processed (from the negative peak) as the whole number of the measurement time processed, where the impedance is equal to zero (for the second moment) (namely, at the beginning of the second peak) for the processed measurement moment where the impedance is equal to zero for the third moment (namely, the end of the second peak) of all the processed differential impedance signal values.
[070] Especially, the comparison of the area under the processed differential signal curve (as defined above) as time-dependent impedance data with reference (known) data for the area under the processed differential signal curve for sperm cells (morphologically) ) normal and (morphologically) abnormal may be based on an implementation of the method for classifying sperm cells.
[071] Here, the method comprises at least (the use of) a pair of electrodes and time-dependent impedance data based on the measurement of at least a first impedance as a function of fluid time and analysis and classification based on time-dependent impedance data. In addition or alternatively, the method may comprise the use of an optical sensor and / or an acoustic sensor to detect a fluid characteristic (comprising a sperm cell) to provide (additional) information for classifying the sperm cell (using optical and / or acoustic). Terms like "sensor" and "device" can also refer to a plurality of sensors or devices, respectively.
[072] In another aspect, the invention also provides a method for analyzing a sperm cell in a fluid, the method comprising: (i) providing a fluid flow comprising the sperm cell to a fluid flow channel, in which the fluid flow channel comprises a first pair of electrodes and optionally a second pair of electrodes; (ii) providing an electrical signal to the first pair of electrodes and optionally to the second pair of electrodes and measuring a first impedance as a function of fluid time (flowing) between the first pair of electrodes and optionally a second impedance as a function fluid time (flowing) between the second pair of electrodes to provide time-dependent impedance data; and (iii) comparison of time-dependent impedance data with predefined reference data in a comparison stage.
[073] In another aspect of the invention, the method, as described herein, is used to improve sperm viability, especially where the reference data contains information on the presence and / or absence of a cytoplasmic gout. In particular, the method can be used to improve the viability of sperm from farm animals, especially pig sperm (wild boar) and cattle sperm (bulls). In additional embodiments, one or more (other) characteristics of the sperm cells are used to improve sperm viability. In particular, reference data may contain information about one or more (other) characteristics.
[074] Consequently, in another aspect, the invention also provides purified sperm from cattle (and pigs) having less than 10% sperm cells with cytoplasmic gout in relation to the total number of sperm cells, especially purified sperm from cattle (and pigs) ) able to be obtained by the method described here. BRIEF DESCRIPTION OF THE DRAWINGS
[075] The realizations of the invention will now be described by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:
[076] Figures 1a-1b schematically depict the system for classifying a sperm cell;
[077] Figure 2 schematically depicts other achievements of the system;
[078] Figures 3a-b schematically depict some aspects of the method for classifying sperm cells;
[079] Figure 4 schematically depicts a differential signal curve;
[080] Figure 5 schematically depicts a model of electrical circuit of an embodiment of the system analysis zone comprising two pairs of electrodes.
[081] Corresponding reference symbols used in the description and in the figures indicate the same or corresponding parts. Schematic drawings are not necessarily scale drawings. DETAILED DESCRIPTION OF ACHIEVEMENTS
[082] The method and system of the invention, as described herein, are based in particular on various functions that can be advantageously combined in different embodiments. The main function, especially, comprises a system and a method to analyze a characteristic of a sperm cell flowing in a fluid channel, especially, to analyze the sperm cell for abnormalities. In particular, the analysis comprises analyzing the impedance measurements made with electrodes provided in the flow channel in an analysis zone, in which the impedance of a fluid flowing comprising the sperm cell over time is used to provide time-dependent impedance data. , for example, comprising (a shape of) an impedance measurement curve. Time-dependent impedance data can be provided using one pair of electrodes as well as using two pairs of electrodes or using additional pairs of electrodes. Here, a shape of an impedance measurement curve can indicate morphological properties (or other characteristics) of the individual cell passing through the electrodes or it can indicate other (particulate) material that passes through the electrodes. In particular, this functionality can be combined with a second functionality, that is, a classification to redirect a sperm cell (abnormal) downstream of the analysis zone, when a specific parameter (characteristic) of that sperm cell, such as an abnormality (morphological), is identified. However, the system and method can also be used to analyze (or identify) only, without performing a classification or separation action. A third feature comprises focusing, in which a sperm cell flowing in the flow channel can be directed to a specific location in the fluid channel, especially to substantially standardize the location of the sperm cell when entering / being present in the analysis zone. Such targeting may, for example, comprise ultrasound, dielectrophoresis, or the use of different liquid flows (hydro-dynamic focusing). The system and method can be used, for example, to identify the presence and / or absence of sperm cells comprising a cytoplasmic drop in which the method is used to improve sperm viability. The use of the system and / or method described here can provide purified sperm (cattle or pig) having less than 10% sperm cells with cytoplasmic gout in relation to the total number of sperm cells.
[083] Figure 1a schematically depicts an embodiment of system 1 for classifying a sperm cell 6 in a fluid 5, according to the invention. The system 1 comprising a fluid flow channel 2 with a first dimension 61 of the flow channel perpendicular to the flow channel axis 63 for carrying the fluid 5, wherein the fluid flow channel 2 comprises an inlet 10, a zone analysis zone 40 downstream of input 10, a classification zone 50 downstream of analysis zone 40, and exits 80, 90, 100, ... configured downstream of classification zone 50. The system comprises at least two exits 80 , 90 (sometimes also mentioned here by a first exit and a second exit), especially to classify between a normal sperm cell 6a and an abnormal sperm cell 6b (for example, comprising a cytoplasmic drop). The system may also advantageously comprise at least one additional outlet 100, shown with dashed lines in Figure 1a. A third outlet 100 can, for example, be used for additional particulate material 8, such as for waste to be directed to it. The routing of an additional particulate material to an additional output 100 may be based on a comparison of the time-dependent impedance data with the predefined reference data. The predefined reference data can be based on data for the additional particulate material 8. The predefined reference data can also be based on data for sperm cells 6. Especially, for this comparison, the time-dependent impedance data can comprise the comparison from data to reference data for sperm cells and determining the absence of sperm cells. The pictured system 1 also comprises an optional focusing zone 20 downstream of the entrance 10 and upstream of the analysis zone 40 and also an optional guidance zone 30 for orienting the sperm cell 6, between the focusing zone (optional) 20 and the analysis zone 40, in which a sperm cell 6 can be oriented, by at least one restriction element 31, as depicted in the embodiment. In other embodiments, the guidance zone 30 may comprise other elements to guide the sperm cell 6. In addition, in additional embodiments, targeting and targeting may be provided in combination in one zone, for example, if targeting is provided by dielectrophoretic forces (see below). The realizations of system 1 can comprise a pair of electrodes 41 or two pairs of electrodes 41, 42, respectively, or even more (pairs of) electrodes, in which a pair of electrodes can comprise exactly two electrodes, but also more than two electrodes , especially, comprising a main electrode and at least one measuring electrode. In embodiments comprising more than one pair of electrodes, the main electrode of one pair of electrodes can also be comprised of more than one pair of electrodes. In an embodiment comprising a first pair of electrodes 41 and a second pair of electrodes 42, for example, the first pair of electrodes may comprise a main electrode and a measurement electrode, and the second pair of electrodes may comprise the same main electrode and another measuring electrode. The embodiment shown in Figure 1a comprises two pairs of electrodes 41 and 42 in flow channel 2, both, comprising respectively a main electrode 41a, 42a and a measurement electrode 41b, 42b. The first pair of electrodes 41 comprises the first intra-electrode distance d1 (between main electrode 41a and measuring electrode 41b) and a second pair of electrode 42 comprising a second intra-electrode distance d2, in which the two pairs of electrodes are configured in an inter-electrode distance D12 (the shortest distance between electrodes 41a and 42a, as well as the shortest distance between electrodes 41b and 42b) distant from each other. In particular, the first intra-electrode distance d1 can be substantially equal to the second intra-electrode distance d2. Especially if the electrodes are configured on the wall of the fluid flow channel, the (first and second) intra-electrode distance d1, d2 can also be equal to the first dimension 61 of the fluid flow channel 2 and / or the second dimension 62 fluid flow channel 2.
[084] In a specific embodiment (not shown, however, which can be explained with the embodiment depicted in Figure 1a), the first pair of electrodes 41 and the second pair of electrodes 42 may comprise a mutual electrode. For example, the first pair of electrodes would comprise a first electrode 41a of the first pair of electrodes 41 being the mutual electrode and a second electrode 41b of the first pair of electrodes 41 and the second pair of electrodes 42 would comprise a first electrode of the second pair of electrodes 42 being the mutual electrode 41a and a second electrode 42b of the second pair of electrodes 42. In this embodiment, the inter-electrode distance D12 is defined as the shortest distance between the electrodes of the two pairs of electrodes, not being the mutual electrode, especially, in this example, the distance being between 41b and 42b.
[085] To perform impedance measurements, an electrical source 140 is connected to the electrode (s) (41 alone or) 41.42 to provide an electrical signal to one of the electrodes 41a, 42a of a pair of electrodes 41, 42 (in embodiments, at least to the first pair of electrodes 41, but, in other embodiments - as in Figure 1a - also, to the second pair of electrodes 42). Also, a measuring device 150 is functionally coupled to the electrodes (41 or) 41, 42 which are provided with the electrical signal to measure an impedance as a function of fluid time 5 (optionally comprising the sperm cell 6) between the pair number of electrodes (depending on the number of electrode pairs (41 or) 41, 42 to measure a first impedance as a function of time or a first impedance as a function of time and a second impedance as a function of time) to provide data of time-dependent impedance. When using only the first pair of electrodes 41, the time-dependent impedance data is based on the impedance measured as a function of time between the first pair of electrodes 41, while the time-dependent impedance data is based on the impedance measured as a time function between the first pair of electrodes 41 and the impedance measured as a function of time between the second pair of electrodes 42 when the system comprises two pairs of electrodes 41, 42.
[086] Terms such as "outlets 80.90, ..." and "outlets 80.90,100 ...", especially, indicate at least two exits, although more are possible, and at least three exits, although more are possible, respectively.
[087] Classification device 51 is specially configured to classify sperm cells 6 by directing sperm cell 6 in classification zone 50 to one of the outputs 80, 90, 100. ... based on a comparison in a comparison stage time-dependent impedance data with predefined reference data. Using the realization given in Figure 1a, the classification can, for example, be made by comparing the time-dependent impedance data (based on the first impedance as a function of time and a second impedance as a function of time) to the data of reference (also based on the reference data of the two pairs of electrodes) to classify sperm cells 6. Classification in classification zone 50 can be provided in different ways by the classification device 51. In one embodiment, the classification device comprises a valve, and the classification can be provided by the valve that controls the flow to one of the outlets 80, 90 (or to one out of one or more optional additional outlets 100). The classification can also comprise dielectrophoretic classification, where an external electric field is applied directly to the sperm cell 6 in the fluid flow 5. In the embodiment depicted in Figure 1a, the classification is provided by an electric field provided by the first electromagnetic device 52, in which sperm cells 6 are directed by dielectrophoretic force. The classification is based on the comparison of time-dependent impedance data with reference data. In particular, the reference data may comprise information about abnormal morphological normal sperm cells 6, including information about sperm cells 6 comprising a cytoplasmic drop, to classify normal 6a sperm cells (morphological) from abnormal sperm cells (morphological) 6b when comparing the data reference with the time-dependent impedance data in the comparison zone 50 in the comparison stage.
[088] Preferably, sperm cells 6 (passing sequentially) are all located in substantially the same location at the time they enter the analysis zone 40. To allow positioning (focusing), especially on the fluid flow channel axis 63, the sperm cells 6 in the focusing zone 20 are specially directed by a focusing device 21 to the fluid flow channel axis 63. The focusing functionality can be provided by dielectrophoretic forces provided by a second electromagnetic device 22, as is depicted in Figure 1a. However, focusing can also be performed by means of hydrofocusing, where system 1 comprises at least one additional inlet configured to provide an additional fluid flow of a support material to the fluid flow channel 2 in the focus zone 20 and the fluid comprising the sperm cells 6 is surrounded by the support material, wherein the fluid comprising the sperm cell 6 is directed to the center of the fluid flow channel 2 (not shown in the figure).
[089] Figure 1b depicts (a top view) a part of an embodiment of system 1, in which the fluid flow channel 2 is rotated 90 ° by the fluid flow channel axis 63 relative to the system 1 pictured (in a side view) in Figure 1a. This realization comprises only a first pair of electrodes 41, of which only one electrode 41a is visible, connected to an electronic device 140 and a measuring device 150 (for illustrative purposes, the connection is pictured although the measuring device does, in fact, connected to measuring electrode 41b (not shown in this figure). The flow channel 2 further comprises an input 10, an analysis zone 40 and a classification zone 50. Outputs 80, 90 (and 100) are not shown in the figure. The second dimension of the flow channel perpendicular to the axis of the fluid flow channel and to the first dimension 61 (not visible) is depicted schematically by reference 62.
[090] Figure 2 schematically depicts some additional aspects of the achievements of system 1 for classifying a sperm cell 6. In Figure 2, an embodiment comprising a fluid flow channel 2 configured on (on) a 1000 chip is depicted. A fluid stream comprising sperm cells 6 can be provided by a pumping device 200. The flow channel 2 comprises two pairs of electrodes 41, 42 for analyzing sperm cell 6 and two electromagnetic devices 22, 52, also shown schematically, as electrodes, although electromagnetic devices 22, 52 may comprise more than one electrode, especially to provide a heterogeneous electric field. In this embodiment, the electrical signal is provided with a main electrode 41a, 42a of the pairs of electrodes 41, 42 by (an electrical source 140) an impedance spectroscope 140, in which a first output channel 142 is connected to the main electrodes 41a and 42a. The impedance spectroscope 140 also functions as the measuring device 150, whereby the measuring electrodes 41b, 42b, of the electrode pairs 41, 42 are connected to a first input channel 151 and a second input channel 152 of the measuring device. measurement 150. In this embodiment, the same electrical signal is provided to the main electrodes 41a and 42a of the first pair of electrodes 41 and the second pair of electrodes 42. Other embodiments can comprise only one main electrode 41a being comprised in the first pair of electrodes 41 and in the second pair of electrodes 42. In particular, the two measurement signals of the two electrodes 41b and 42b, in this embodiment, are both classically amplified with a pre-amplifier 155. For measurements in the differential state, the absolute impedance data of the second electrode pair 42 are subtracted from the signal of the first electrode pair 41 prior to peak detection and storage. An optional control system 300 is also pictured, in which the control system can control the pumping device 200 and, if relevant, the sorting device (via an electrical source 140) and a focusing device 21 (not pictured) ). The control system 300 can also be applied to process the measured signal and the control system 300 can also comprise options for graphically presenting the analysis. In the figure, too, the achievements of the focusing device 21 and the sorting device 51 are depicted. In particular, the focusing device is configured as a second electromagnetic device 22, in which the focusing functionality in a sperm cell 6 (not shown) is provided by an electric field provided by the second electromagnetic device 22 which is connected to a generator in a specific way. waveform 120. The classification device 51 is configured as a first electromagnetic device 52, in which a sperm cell 6 can be directed to one of the outlets 80, 90 (to a first container 85 or to a second container 95) based on the identification in the analysis zone by means of an electric field provided by the first electromagnetic device 52 which is connected to the second output channel 141 of the electronic source 140. In particular, the classification device can be arranged in the fluid flow channel 2, as pictured in Figure 1a. In a specific embodiment, the sorting device, especially comprising the first electromagnetic device 52 is configured outside the fluid flow channel 2. Especially, also, the focusing device 21 can be arranged in the fluid flow channel 2. In a specific embodiment, the focusing device 21, especially comprising the second electromagnetic device 22 is configured outside the fluid flow channel 2.
[091] In Figure 2, a system is pictured schematically using two pairs of electrodes 41, 42 and explained above for measurement in a differential state. System 1, however, can also be used, applying only the first pair of electrodes 41 (and disconnecting the second pair of electrodes 42) (likewise, an embodiment of system 1 comprising only the first set of electrodes 41 can be applied ) and measurement in a non-differential state. In particular, for this purpose, a 4-point measurement can be performed, in which the first electrode 41a of the first pair of electrodes is connected to the first output channel 142 and the second input channel 152, while the second electrode 41b of the first pair of electrodes is connected to the first electrode 41a of the first pair of electrodes. electrodes are connected directly to the second input channel 142 and, at the same time, the second electrode 41b of the first pair of electrodes is connected via the preamplifier 155 to the first input channel 151 of the measuring device 150. This configuration allows you to measure differential voltage on the second input channel 152 when connecting the first and second electrodes to the second input channel 152 and measure the amplified current when connecting the first electrode 41a to the first output channel 142 and the second electrode 41b to the first channel input 151 (via the preamp 155). All other connections can be maintained, as described above. In fact, a non-differential measurement can also be performed by a 2-point measurement, in which (only) the current between electrodes 41a, 41b of a pair of electrodes 41 is measured and the voltage is adjusted (by connecting the output 142 to the main electrode 41a, and the input channel 151 via the preamplifier 155 to the measurement electrode 41b).
[092] In Figures 3a and 3b, some typical examples (represented graphically) are depicted for a measurement curve of a normal sperm cell (Figure 3a) and a sperm cell comprising a cytoplasmic drop (Figure 3b). In the figures, differential measurement data are generally depicted, showing a first positive peak A and a second negative peak B. In particular, the drop can be identified due to providing an extra D edge on the peaks, graphically, more pronounced on the second peak (negative ) B.
[093] Figure 4 presents an accomplishment to analyze the time-dependent impedance data, especially here, also provided (graphical representation of) by the differential signal curve data, showing a first positive peak A and a second negative peak B The classification of sperm cells 6 can be based on the area under the processed measurement curve, especially below the negative peak B when comparing said area with the reference data of the area under the (processed) curve for normal morphological sperm cells and abnormal morphological sperm cells. The area under the processed curve can be provided, first, by normalizing the measurement curve based on the height of peak YB and the width of peak XB. Especially, after normalizing the curve, a significant difference was found for an area under the processed measurement curve caused by an abnormal sperm cell 6 (comprising the cytoplasmic drop) and an area under the processed measurement curve caused by a normal sperm cell 6 .
[094] Figure 5 schematically depicts an Electrical Circuit Model (ECM) of an embodiment of analysis zone 40 comprising two pairs of electrodes for differential impedance analysis. Without a cell 6 between the pair of electrodes 41, 42 (pictured in the second pair of electrodes 42), system 1 is described by an electrode-electrolyte interface (double layer) (Cdl), electrolyte (comprising a Rel resistance and a capacity Cel) of fluid 5, the parasitic effects of microelectrodes (Cpar) and wire resistance (Rw). A passing sperm cell 6 adds a cell membrane capacitance (Cmem) and cytoplasm resistance (Rcyto) to the ECM, considering Foster and Schwan's simplified ECM for a single protection spheroid in suspension (pictured on the first electrode 41). EXPERIMENTAL Materials and methods Chip Manufacturing
[095] Microfluidic chips were manufactured using water-based photolithography, blasting and bonding techniques. After cleaning, two borofloat wafer disks (BF33, 100 mm in diameter, 500 and 1100 μm thick), microelectrodes were manufactured after deposition of resistance, exposure and development, engraving with BHF water, deposition of titanium layers / platinum (layer thickness of 30 and 120 nm, respectively) and elevation of resistance. Subsequently, inserts for fluidic and electrical connections were powder blasted through both wafer-type discs (particle size 30μm). After cleaning the wafer-type discs using ultrasound and HNO3, a plate layer (20 μm, PerMX3020, Dupont) was laminated on the 500 μm wafer discs at 80 ° C. After lamination, the wafer-type discs were precooked (5 min at 50 ° C, 5 min at 65 ° C and 10 min at 85 ° C) to improve the adhesion of the plate to the glass. The exposure was carried out using a 12mW / cm2 UV source. Subsequently, a post-exposure cooking was carried out (5 min at 50 ° C, 5 min at 65 ° C and 10 min at 85 ° C). The polymer layer was developed using a spin coating. After aligning the 500 μm wafer discs in relation to the 1100 μm wafer discs using a link fixation, they were linked using an anodic fixer. Subsequently, the stack of wafer-type discs was cooked to harden in a heated press. After dicing, the chips were ready to use. Two different chip designs were used in the experiments described. For the electrical analysis of sperm morphology, differential impedance measurements were performed on a 20 μm high and 20 μm wide channel containing two pairs of electrodes with an electrode width of 10 μm and a rating of 20 or 40 μm. Impedance-based cell classification experiments were performed on a 20 μm high and 100 μm wide channel using a single pair of electrodes with a width of 20 or 50 μm. Sample and chip preparation
[096] Fresh boar semen was obtained from a local artificial insemination center at a concentration of 20 x 106 ml-1 cells. The samples were diluted with Beltsville Thawing Solution (BTS) to a concentration of 5 x 106 ml-1 cells. Before each experiment, the microfluidic channel was coated with poly (L-lysine)-grafted-poly (ethylene glycol) (PLL-g-PEG) to prevent cell adhesion. PLL-g-PEG was rinsed through the channel at a concentration of 100 μg ml-1 in DI water for at least 15 min. at a flow rate of 0.5-1 μl / min using a syringe pump. BTS solution was rinsed for at least 15 min. at a flow rate of 0.5-1 μl / min to remove the remaining coating solution. Subsequently, sperm solution was flowed through the channel at a flow rate of 0.5-1 μl / min. Through the visualization in the microfluidic channel, the flow rate was changed to 0.013 - 0.75 μl / min before the impedance acquisition. Detection and Impedance Analysis
[097] The impedance was recorded using a Zurich HF2IS impedance spectroscope equipped with an HF2TA preamplifier (also shown in Figure 2). Two different modes of operation were used in the experiments. In the differential state, an AC signal with an amplitude of 0.5 V was generated at output 1 and applied to the differential electrode pair of the devices under test (DUT). The two corresponding electrodes of the differential electrode pair were connected to input 1 and input 2 of the impedance spectroscope through two channels of classified current amplification of the pre-amp. HF2TA. In the non-differential state, a 4-point measurement was performed. The current was amplified using channel 1 of the HF2TA current amplifier to input 1 of the impedance spectroscope. The voltage was measured differently at input 2. In both states, the impedance was recorded using a sinusoidal excitation of 1 MHz with an amplitude of 0.5 V, a bandwidth of 200 Hz and a sample frequency of 3598 Hz, unless mentioned otherwise. The recorded impedance data was imported and processed in Matlab (R2013a, Math Works). For measurements in the differential state, the absolute impedance data from input 2 was subtracted from signal 1 prior to peak detection and storage. In the non-differential state, deviation and compensation were removed when using a moving average filter. Subsequently, peaks were detected and classified. Cell focusing and classification using dielectrophoresis
[098] The orientation and location of the sperm cell within the microchannels were processed using dielectrophoresis (DEP). Cellular focusing was performed by applying a 10 MHz, 6VPp sinusoidal excitation to the focusing electrodes (Agilent X) unless otherwise noted. Similarly, cell classification by identical excitation using Aux1 output from the impedance spectroscope. Image analysis
[099] Sperm tracking was performed using the “motion based multiple object tracking” function of the Matlab vision computer system tools. This function processes each structure one by one and detects objects by comparison to a static base. These objects are tracked over time and assigned to object paths. This function readily available in Matlab has been adapted to allow storage of time data, location and sizes of objects. To investigate the effect of sperm location and size on impedance, these data were combined with acquired impedance data. Integrated DEP data acquisition and classification using LabVIEW
[100] Classification of sperm cells using DEP based on impedance data requires a control system that combines both techniques. In addition, this system should allow control by the syringe pump and the acquisition of optical data for verification purposes. Fortunately, virtual instrument (VI) actuators are available for all equipment involved. These triggers take care of the low-level communication between the computer (LabVIEW) and the instruments, and contain high-level functions to control them. Upon initialization of the LabVIEW control program, the impedance spectroscope, pump and camera are initialized when selected in the user interface. After configuring the instruments and starting the experiment, the experimental data (video and impedance) and instrument reports are automatically saved. Once the video and impedance measurements are timed within the program, the corresponding data files are synchronized. In the classification mode, the control program monitors the impedance over time. Through the passage of the particle or cell, there is a change in impedance. Simulation
[101] The electrical response of the microfluidic configuration was investigated when building a numerical model of the circuit in Matlab. This model is well described in the literature and is based on the simplified electrical circuit (ECM) model by Foster and Schwan for a single protection spheroid in the suspension. In the simulations, a parallel electrode configuration was modeled without a field impression at the electrode edges. In addition, sperm cells were modeled as spheroids with equal cell volume (1.21x10-15 m3). Results of Electrical circuit model
[102] Impedance spectroscopy is a tool commonly used for label-free analysis of adherent cells or cells in suspension. This technique has been used extensively to investigate the dielectric properties of cells in microfluidic systems. The construction of an electrical circuit model (ECM) is a simple way to obtain knowledge of the electrical response of the microfluidic configuration (figure 5). The capacitive properties of the microelectrode configuration are predominantly determined by the electrode / electrolyte interface (Cdl), the electrolyte (Cel) and the parasitic effects of the microelectrodes (Cpar). The resistive response is influenced by the conducting wires (Rw) and the conductivity of the electrolyte (Rel). When a sperm cell is introduced between the microelectrodes, the capacitive and resistive properties will be altered by the cell membrane (Cmem) and the cell's cytoplasm (Rcyt), respectively. The simulation showed a great effect of the double layer on the absolute impedance. Due to a small electrode surface area and a correspondingly small Cdl, the impedance decreased continuously over a wide frequency range. At a frequency of approximately 1.3 MHz, a resistive level was formed. A frequency scan of the electrode configuration showed similar behavior compared to the simulation, indicating that a measurement frequency of 1.3 MHz is an adequate choice for analyzing sperm impedance in this configuration. At this frequency, the simulation showed an impedance increase of approximately 800 Q when introducing a sperm cell between the electrodes. Orientation impedance analysis and cell morphology
[103] Impedance analysis was performed by flowing sperm cells through a microfluidic channel 20 μm high, 100 μm wide with a channel restriction of 20 μm wide at a flow rate between 0.013 and 0.02 μl / min -1. The impedance was recorded differently with two pairs of electrodes with an electrode width of 10 μm and an electrode classification of 20. After calculating the difference between the electrical responses of both pairs of electrodes, baseline correction and peak detection were carried out. The resulting peak height distribution was in good agreement with the simulated impedance change in the case of a single sperm passing through the electrodes. However, this distribution showed a great dispersion in the data, ranging from values between 200 and 2500 Q. Factors that influence the amplitude of this distribution are, for example, the orientation, location of the cell and cellular properties. Due to these factors, the change in absolute impedance is not an adequate parameter to characterize morphological differences. A different approach is to analyze the peak shape over time. A sperm cell has a very distinctive shape and its typical length is greater compared to the microchannel geometries (width and length) and the width of the microelectrodes. When a sperm cell is flowed through this microchannel, the cell will align itself along its longitudinal axis in relation to the channel wall. Consequently, the different parts of the sperm cell (head, middle part and flagellum) will pass the electric field between the microelectrodes at different points in time and will affect the recorded impedance in the same way. As a result, the peak shape may contain information about cell orientation and its morphology.
[104] To test this hypothesis, the peak impedance form of the passing sperm cells was investigated (using an electrode rating of 20 μm). The spectra showed a positive and negative peak (figure 3), corresponding to the sperm passing through the first and second pair of electrodes, respectively. At zero crossover, the impedance recorded at input 1 and 2 is equal, at which point, the sperm head is positioned between the two pairs of electrodes, approximately. The spectra showed a clear peak-oriented cell orientation effect. When a sperm passes the electrodes first with the head, the impedance recorded over time showed a positive peak, a negative peak and a slight difference in impedance before returning to zero the last corresponding to the presence of the sperm flagellum between the electrodes. In the orientation of the tail first, this small difference in impedance was observed before the sperm head reached the first pair of electrodes. In addition, information about cell morphology could be extracted from the data. Content of cytoplasmic gout in the flagellum resulted in the amplification of the measured peaks. A clear example is illustrated in Figure 3b, in which a clear protrusion in the signal is observed between the minimum peak and the small impedance change corresponding to the sperm flagellum.
[105] One way to extract information regarding the cytoplasmic drop content from data impedance data is to analyze the area under the curve (AUC). In total, 18 morphologically normal sperm cells and 18 containing gout were selected for analysis. Using Matlab, the maximum point (figure 4, A), minimum point (B) and crossing zero (C) were determined. Subsequently, the AUC of the positive and negative peak was calculated. When comparing the AUC averages of the negative peaks of the two populations using a t-test (paired sample), no statistical difference was found (p = 0.52). A plausible explanation is the effect of the cell's orientation, location and speed on the AUC. The orientation (cell slope) and location influence at the height of the peak and the speed of the cell have an effect on the amplitude of the peak. After correcting the peak height (YB) and peak amplitude (XB), a significant difference is found between the AUCs of both populations (p = 0.003), see the following table:
Effect of dielectrophoretic focusing on cell location and speed
[106] The location and speed of the cell are important parameters to control the design of a cell classification system. The defined cell location and speed are necessary to make accurate measurements of sperm morphology and to control cell classification after analysis. Dielectrophoretic focusing is used to control these parameters. To present the effect of DEP focusing on cell location, sperm cells were flowed through the microfluidic channel with and without DEP excitation. Without DEP excitation, the distribution of sperm cells within the channel is random. With DEP excitation, the sperm cells were clearly deflected in the middle of the channel, which is confirmed by a small distribution of the Y location. The speed of the sperm cells was investigated close to the impedance electrodes. The location and speed of the cell were determined shortly after the passage of the 20 μm electrode pair. The impedance data was combined with the video data to investigate the effect of speed, orientation and location. First of all, the speed and location of the cell were investigated with and without DEP targeting. Without focusing, the image analysis showed wide distributions in the location and speed of the cell (the middle of the channel was positioned at approximately 64 μm; the edges of the channel are positioned at approximately 12 and 116 μm). With focusing, the range of these distributions has been reduced extensively, as can be seen especially from minor differences between the median value and the first and third quartile values (that is, distance between quartiles) found after focusing compared to the difference observed without focusing , see table below: It is observed that there were no significant effects of the average cell speed and location on the recorded impedance.
Sperm cell impedance controlled classification
[107] The next step in the development of a free tagging cell classification system is the design of an algorithm that is able to actively classify sperm cells based on impedance detection. As a proof-of-concept experiment, the beads and sperm cells were classified based on impedance. Lab VIEW was chosen as a development platform. After targeting and detection, beads and sperm cells must be actively classified. The LabVIEW program continuously monitors the impedance. Whenever a change in impedance is recorded, of which the peak shape corresponds to the peak model, the width and height of the peak are determined. The peak width is used to calculate the particle speed in order to predict the estimated time of arrival (ETA) on the classification electrodes. The total peak height determines whether a particle is classified or not. This selection is based on the impedance window of interest (WOI). In this example, 3 μm polystyrene beads will be separated from the sperm cells. A mixture of sperm cells and beads was flowed through the microfluidic channel at a flow rate of approximately 0.025 μl / min-1. The impedance WOI was set to 4-8 Ohm, which corresponds to the change in impedance when a bead passes through the electrodes. The average impedance change in sperm cells is approximately 17 Ohms, which is above WOI. Whenever a particle's impedance change is detected, which fits within WOI, DEP electrodes are activated to classify the particle in the upper channel. When the beads pass through the electrodes, impedance peaks were registered within WOI, consequently, the classification of the beads actively in the upper channel in the channel division. Whenever sperm cells or waste pass the detection electrodes, the recorded impedance was above or below the WOI. As a result, sperm cells and waste were collected in the lower channel without being deflected by the classification electrodes. The classification speed in the described experiment was low (<1 s-1 sperm cell) due to the low concentration of beads and sperm and small flows. In addition, the classification speed of this system is limited to approximately 5 s-1 cells due to the limitations in the computational speed of the LabVIEW software. However, the classification was approximately 100% effective.
[108] The term "substantially" here, as in "consists substantially", will be understood by the person skilled in the art. The term "substantially" can also include achievements with "entirely", "completely", "everything" etc. Then, in the realizations, the adverb can substantially also be removed. When applicable, the term "substantially" may also refer to 90% or more, such as 95% or more, especially 99% or more, even more especially 99.5% or more, including 100%. The term "comprise" also includes accomplishments where the term "comprises" means "consists of". The term "and / or" especially refers to one or more of the items mentioned before and after "and / or". For example, a phrase “item 1 and / or item 2” and similar phrases may refer to one or more of item 1 and item 2. The term “comprising” may, in one embodiment, refer to “consisting of ”, But may, in another embodiment, also refer to“ containing at least the defined species and, optionally, one or more other species ”.
[109] In addition, the terms first, second, third and similar in the description and in the claims are used to differentiate between similar elements and not necessarily to describe a sequential or chronological order. It is to be understood that the terms thus used are interchangeable under suitable circumstances and, in those embodiments of the invention described here, are capable of operation in sequences other than those described or illustrated here.
[110] The devices here are, among others, described during the operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation or devices in operation. It should be noted that the achievements mentioned above illustrate or rather limit the invention, and that those skilled in the art will be able to design many alternative embodiments without deviating from the scope of the appended claims. In claims, any reference signs placed in parentheses should not be construed as limiting the claim.
[111] The use of the verb “to understand” and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article “o / a” or “um / uma” preceding an element does not exclude the presence of a plurality of these elements.
[112] The invention can be implemented by means of hardware comprising several different elements, and by means of a properly programmed computer. In the device claim enumerating several means, several of these means can be incorporated by one and the same piece of hardware. The mere fact that certain measures are mentioned in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.
[113] The invention still applies to a device comprising one or more of the characterizing aspects described in the description and / or presented in the accompanying drawings.
[114] The invention still belongs to a method or process comprising one or more of the characterizing aspects described in the description and / or presented in the accompanying drawings.
[115] The various aspects discussed in this patent can be combined in order to provide additional advantages. Still, the technician in the subject will understand that the achievements can be combined, and that more than two achievements can also be combined. In addition, some aspects may form the basis for one or more division orders.

权利要求:
Claims (17)
[0001]
1. SYSTEM (1) FOR PERFORMING ANALYSIS AND SELECTION OF SPERM, based on the sperm cell morphology of the sperm cells (6) in a fluid (5), the system (1) being characterized by comprising: (i) a channel of fluid flow (2) for carrying said fluid (5), the fluid flow channel (2) comprising an inlet (10), an analysis zone (40) configured downstream of said inlet (10) and comprising a first pair of electrodes (41) comprising a first intra-electrode distance (d1) and a second pair of electrodes (42) comprising a second intra-electrode distance (d2) and configured at an inter-electrode distance (D12) from the first pair electrodes (41), a classification zone (50) configured downstream of said analysis zone (40), and outputs (80.90, ...) configured downstream of said classification zone (50); (ii) an electrical source (140) configured to provide an electrical signal to the first pair of electrodes (41) and an electrical signal to the second pair of electrodes (42); (iii) a measuring device (150) operably coupled to the first electrode pair (41) and operatively coupled to the second electrode pair (42), and configured to measure a first impedance as a function of fluid time (5) between the first pair of electrodes (41) and to measure a second impedance as a function of fluid time (5) between the second pair of electrodes (42) and to provide time-dependent impedance data based on first impedance and second impedance, where time-dependent impedance data comprises differential signal curve data, where differential signal curve data is provided by subtracting a second impedance as a function of time from the first impedance as a function of said time; (iv) a sorting device (51) configured to classify sperm cells (6) between normal morphological sperm cells and abnormal morphological sperm cells by directing the sperm cell (6) in the classification zone (50) to one of the exits (80, 90, ...) based on a comparison in a comparison stage of the time-dependent impedance data with predefined reference data, in which in the comparison stage, a differential signal curve of the differential signal curve data is compared with a differential signal curve of normal sperm cell differential signal curve and a differential signal curve of abnormal sperm cell differential signal curve.
[0002]
2. SYSTEM, according to claim 1, characterized in that, in the comparison stage: (i) a shape of the differential signal curve of signal curve data is compared with a shape of the differential signal curve of signal curve data differential signal from normal sperm cells and a form of the differential signal curve from abnormal sperm cell differential signal curve, where the differential signal curve from normal sperm cell differential signal curve shows a positive and negative peak , and where the differential signal curve of abnormal sperm cell differential signal curve an additional peak between the negative peak and the negative peak widening; or (ii) an area under the differential signal curve of normal sperm cells and an area under the differential signal curve of abnormal sperm cells.
[0003]
SYSTEM, according to any one of claims 1 to 2, in which the fluid (5) is characterized by still comprising an additional particulate material (8), in which the system comprises an additional outlet (100), and in which the classification device (51) is further configured to classify the additional particulate material (8) by directing the additional particulate material (8) in the classification zone (50) to one of the exits (80,90,100, ...) based in a comparison in the time-dependent impedance data comparison stage with predefined reference data.
[0004]
4. SYSTEM, according to any one of claims 1 to 3, characterized in that it also comprises a focusing zone (20) configured upstream of said analysis zone (40) and downstream of said entrance (10), and a second electromagnetic device (22) to provide an electric field to the focusing zone (20), wherein the classification device (51) comprises a first electromagnetic device (52) to provide an electric field to the classification zone (50), and the the first electromagnetic device (52) is configured to direct the sperm cell (6) by dielectrophoretic force to one of the outputs (80, 90, ..), and the second electromagnetic device (22) is configured to direct the sperm cell (6) in the focusing zone by dielectrophoretic force.
[0005]
A system according to any one of claims 1 to 4, characterized in that a first dimension (61) selected from the height and width of the fluid flow channel (2) is selected from the range of 5 - 400 μm and a second dimension (62) selected from the width and height of the fluid flow channel to be selected from the range of 5 - 400 μm, and in which a cross-sectional area is selected from the range of 100 - 10,000 μm2.
[0006]
6. SYSTEM according to any one of claims 1 to 5, characterized in that the reference data contains information about the presence or absence of a cytoplasmic drop.
[0007]
7. SYSTEM according to any one of claims 1 to 6, characterized in that the inter-electrode distance (D12) is selected from the range of 10 - 100 μm and the time-dependent impedance data is based on a difference between an absolute value of the first impedance at one moment and an absolute value of the second impedance at that moment.
[0008]
SYSTEM, according to any one of claims 1 to 7, characterized in that (i) the first pair of electrodes (41) is configured on two opposite sides of the flow channel (2), with the flow channel (2) between them, and (ii) the second pair of electrodes (42) is configured on two opposite sides of the flow channel (2), with the flow channel (2) between them, in which the first intra-electrode distance (d1 ) and the second intra-electrode distance (d2) are substantially the same.
[0009]
9. SYSTEM, according to any one of claims 1 to 8, characterized by the sorting device (51) directing an abnormal sperm cell to one of the outlets (80, 90, ...) and a normal sperm cell to another outlet ( 80, 90, ...).
[0010]
10. METHOD FOR CLASSIFICATION OF SPERM CELLS (6) in a fluid (5) between normal morphological sperm cells and abnormal morphological sperm cells, the method being characterized by comprising: (i) provision of a fluid flow comprising sperm cells (6 ) to a fluid flow channel (2), wherein the fluid flow channel (2) comprises a first pair of electrodes (41) and a second pair of electrodes (42) comprising a second intra-electrode distance (d2 ) and configured at an inter-electrode distance (D12) from the first pair of electrodes (41); (ii) providing a first electrical signal to the first pair of electrodes (41) and providing a second electrical signal to the second pair of electrodes (42); (iii) measuring a first impedance as a function of fluid time (5) between the first pair of electrodes (41) and measuring a second impedance as a function of fluid time (5) between the second pair of electrodes ( 42) and to provide time-dependent impedance data based on the first and second impedance, where time-dependent impedance data comprises differential signal curve data, where differential signal curve data is provided by subtracting a second impedance as a function of time from the first impedance as a function of said time; (iv) classification of sperm cells (6) between normal morphological sperm cells and abnormal morphological sperm cells based on the comparison of time-dependent impedance data with predefined reference data in a comparison stage, where in the comparison stage, a differential signal curve of the differential signal curve data is compared with a differential signal curve of normal sperm cell differential signal curve and a differential signal curve of abnormal sperm cell differential signal curve data.
[0011]
11. METHOD according to claim 10, characterized in that, in the comparison stage: (i) a shape of the differential signal curve of signal curve data is compared with a shape of the differential signal curve of data curve differential signal from normal sperm cells and a form of the differential signal curve from abnormal sperm cell differential signal curve, where the differential signal curve from normal sperm cell differential signal curve shows a positive and negative peak , and where the differential signal curve of abnormal sperm cell differential signal curve an additional peak between the negative peak and the negative peak widening; or (ii) an area under the differential signal curve of normal sperm cells and an area under the differential signal curve of abnormal sperm cells.
[0012]
12. METHOD, according to claim 10, characterized by comparing, in the comparison stage, comprising comparing an area processed under the differential signal curve with reference data of an area processed under the differential signal curve for normal morphological sperm cells and for abnormal morphological sperm cells, where the areas processed under the curves are provided by normalizing the respective differential signal curve based on a peak height (YB) and a peak width (XB), where the peak height (YB) is a minimum impedance signal value of a negative peak of the respective curve and the peak width (XB) is the corresponding measurement time at the peak height (YB) minus the measurement time corresponding to a peak start negative.
[0013]
13. METHOD according to any one of claims 10 to 12, characterized in that it further comprises focusing the sperm cell (6) on the fluid flow channel (2) at a location upstream of the first pair of electrodes (41) and wherein focusing the sperm cell (6) comprises (i) providing an additional fluid flow of a support material to the fluid flow channel (2) to surround the sperm cell (6) with the additional fluid flow or (ii) providing a non-uniform electric field to the sperm cell (6) to direct the sperm cell (6) in the fluid flow.
[0014]
14. METHOD, according to any one of claims 10 to 13, in which the impedance measurement is characterized by comprising the measurement of the impedance at a selected frequency in the range of 10 kHz - 100 MHz.
[0015]
15. METHOD according to any one of claims 10 to 14, characterized by the classification of sperm cells (6) based on the comparison of time-dependent impedance data with predefined reference data including the comparison of time-dependent impedance data with a symmetrical curve.
[0016]
16. METHOD according to any one of claims 16 to 15, wherein the fluid (5) is characterized by comprising sperm cells (6) in a concentration of 2 »103 - 2» 108 cells / ml and in which the sperm cells are selected from the group of cattle sperm cells consisting of boar sperm cells (6) and bull sperm cells.
[0017]
17. METHOD according to any one of claims 10 to 16, characterized in that, during selection, an abnormal sperm cell is directed to one of the outlets and a normal sperm cell is directed to another outlet.

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法律状态:
2020-07-28| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-13| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/09/2016, OBSERVADAS AS CONDICOES LEGAIS. |

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