Abstract
A motile cell sorting device is disclosed. The device comprises a chamber, an inlet and an outlet in fluid communication with the chamber, and a plurality of discrete barriers disposed in the chamber. Each discrete barrier comprises at least one wall and at least one acute edge orientated towards the outlet.
Claims
1. A motile cell sorting device comprising: a chamber; an inlet and an outlet in fluid communication with the chamber; and a plurality of discrete barriers disposed in the chamber, wherein each discrete barrier comprises at least one wall and at least one acute edge orientated towards the outlet.
2. The device of claim 1, wherein the at least one wall comprises: first and second walls; and wherein the at least one acute edge comprises: a first acute edge between the first and second walls.
3. The device of claim 2, wherein the first wall is convex, straight or concave.
4. The device of claim 2, wherein the second wall is concave.
5. The device of claim 2, wherein the second wall is straight.
6. The device of claim 2, wherein the at least one acute edge further comprises: a second acute edge.
7. The device of claim 6, wherein the second acute edge is between the first wall and the second wall.
8. The device of claim 7, wherein the at least one wall comprises: a third wall; wherein the second acute edge is between the second and third walls.
9. The device of claim 1, wherein the chamber comprises a channel running between the inlet and the outlet provided at first and second ends respectively and comprising first and second channel walls.
10. The device of claim 1, wherein the chamber is disk-shaped having a periphery and a centre and wherein the inlet is annular and arranged around the periphery of the chamber and the outlet is arranged at the centre.
11. The device of claim 1, wherein the chamber has a height which is between 50 to 300 μm.
12. The device of claim 1, wherein the discrete barriers have a width of between 10 and 500 μm.
13. The device of claim 1, wherein the discrete barriers have a length of between 10 and 1,000 μm.
14. The device of claim 1, wherein the discrete barriers are separated from neighbours in a first direction by a first gap of between 10 to 500 μm.
15. The device of claim 1, wherein the discrete barriers are separated from neighbours in a second different direction by a second gap of between 10 to 500 μm.
16. The device of claim 1, wherein the discrete barriers project into the chamber from a floor or a ceiling.
17. The device of claim 1, wherein the discrete barriers have the same shape and/or the same dimensions.
18. The device of claim 1, wherein the discrete barriers are arranged in a periodic array.
19. The device of claim 18, wherein the array is a rectangular array.
20. The device of claim 18, wherein the array is a hexagonal array.
21. An intrauterine insemination kit comprising a motile cell sorting device, the motile cell sorting device comprising: a chamber; an inlet and an outlet in fluid communication with the chamber; and a plurality of discrete barriers disposed in the chamber, wherein each discrete barrier comprises at least one wall and at least one acute edge orientated towards the outlet.
22. A method of using a motile cell sorting device, the motile cell sorting device comprising: a chamber; an inlet and an outlet in fluid communication with the chamber; and a plurality of discrete barriers disposed in the chamber, wherein each discrete barrier comprises at least one wall and at least one acute edge orientated towards the outlet, the method comprising: supplying a sample comprising motile cells to the inlet; waiting for a period of time of at least 1 minute; and after waiting for the period of time, collecting a refined sample from the outlet.
23. The method of claim 22, wherein the period of time is at least 5 minutes.
24. The method of claim 22, wherein the period of time is between 10 to 60 minutes.
25. The method of claim 22, further comprising: causing the device to be heated to a temperature for incubation.
26. The method of claim 25, wherein the temperature is 37° C.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Certain embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:
[0035] FIG. 1 is a perspective view of a microfluidic chip having first and second ports and a microchannel running between the first and second posts and containing entraining structures;
[0036] FIG. 2 is a perspective view of an array of entraining structures;
[0037] FIG. 3 is a plan view of a first type of entraining structure;
[0038] FIG. 4 is a second type of entraining structure;
[0039] FIG. 5 is a plan view of a microchannel containing an array of the first type of entraining structure;
[0040] FIG. 6 is a plan view of a microchannel containing an array of the second type of entraining structure;
[0041] FIGS. 7a and 7b are plan views of further examples of entraining structure;
[0042] FIG. 8 is a plan view of yet another example of an entraining structure;
[0043] FIGS. 9a to 9c is a plan view of wall structures;
[0044] FIG. 10a are photographs of a first device and the entraining structures;
[0045] FIG. 10b are photographs of a fisecondrst device and the entraining structures;
[0046] FIG. 11 is a process flow diagram of a method of sorting and extracting spermatozoa;
[0047] FIG. 12 is a process flow diagram of a method of sorting and extracting spermatozoa;
[0048] FIG. 13 is a table of results of sample analysis;
[0049] FIG. 14 is a table of results of sample analysis;
[0050] FIG. 15 is a table of results of sample analysis;
[0051] FIG. 16 is a table of results of sample analysis;
[0052] FIG. 17 is a table of summary results of sample analysis;
[0053] FIG. 18 is a table of summary results of sample analysis;
[0054] FIG. 19 is a bar chart of spermatozoa motility;
[0055] FIG. 20 is a bar chart of spermatozoa motility;
[0056] FIG. 21 is a bar chart of spermatozoa morphology;
[0057] FIG. 22 is a bar chart of spermatozoa morphology;
[0058] FIG. 23 is a bar chart of spermatozoa DNA fragmentation;
[0059] FIG. 24 is a bar chart of spermatozoa DNA fragmentation;
[0060] FIG. 25 are photographs of spermatozoa DNA fragmentation;
[0061] FIG. 26 are photographs of spermatozoa DNA fragmentation.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0062] Referring to FIG. 1, a device 1 for sorting motile cells 2, such as spermatozoa, in a sample 3 is shown.
[0063] The device 1 includes a chamber 4, for example in the form of a low-height channel or disc, an inlet 5, and an outlet 6 in fluid communication with the channel 4. The chamber 4 has a height which is less than, preferably much less than (at least by a factor 10 or even 100) its lateral dimensions, such a length and width. The chamber 4 has a height, h, which is preferably between 50 to 300 μm. The chamber 4, inlet 5 and outlet 6 are arranged such that when a sample 3 containing motile cells 2 is supplied to the inlet 5, motile cells 2 swim through the chamber 4 towards the outlet 6. As they swim through the chamber 4, the motile cells 2 are sorted and separated on the basis of motility such that less motile cells 2 (e.g., immotile cells) tend to be retained in the chamber 4, while more motile cells 2 tend to progress along the chamber 4.
[0064] Referring also to FIG. 2, the device 1 includes an arrangement 7 of discrete barriers 8 (or “ratchet”) disposed in the chamber 4 projecting into the chamber 4 from a first inner surface 9, e.g., the floor of the chamber 4, to a second, opposite inner surface, e.g., its ceiling. The arrangement 7 preferably takes the form of a periodic two-dimensional array, such as a hexagonal or cubic lattice. The barriers 8 are separated from side neighbours by a first gap, g.sub.1, of between 10 to 500 μm and from neighbours in front and behind by a second gap, g.sub.2, of between 10 to 500 μm
[0065] Each barrier 8 is generally asymmetrical having differently-shaped first and second faces 13, 14 orientated towards and away from the first port 5 respectively. The first face 13 includes at least one wall 15 and the second face 14 preferably includes at least one wall 16. Herein, the walls 15, 16 may be referred to as “side walls”. The walls 15, 16 or opposite ends of the wall 15 meet at one or more acute edges 17 (herein referred to as “discontinuities”). The angle between the walls 15, 16 is greater than 0° and less than 90° and is preferably less than 30°. The curvature of the edge 17 is greater than 0 μm and less than or equal to 50 μm, micron and is preferably less than 20 μm.
[0066] The chamber 4, inlet 5, outlet 6 and barriers 8 are configured such that motile cells 2 take a time, t, of between 10 to 60 minutes, preferably about 20 minutes, to swim from the inlet 5 through the chamber 4.
[0067] The device 1 may operate at ambient temperature, i.e., room temperature. However, the device 1 may be provided with a heater (not shown), for example in the form of hot plate, oven or water bath, to elevate the operating temperature of the device to a suitable temperature for incubation, for example, about 37° C.
[0068] Referring in particular to FIG. 1, the device 1 preferably takes the form of a microfluidic chip. The device 1 may comprise an assembly of first and second planar portions (not shown) formed from glass and/or or polymeric materials, such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA) or poly octanediol-co-citrate (POC). The first portion (which may be referred to as a “base”) may have a patterned face (not shown) defining a bottom (or “floor”) and sides of the chamber 4 and the barriers 8. The second portion (which may be referred to as a “cover”) may be featureless (e.g., flat) and may define a top (or “ceiling”) of the chamber 4. The cover may also include first and second ports (not shown) an unrefined sample and a refined sample may be provided and collected respectively. The first and second portions (not shown) may consist of the same material or different materials. The device 1 may be fabricating in different ways, for example, by moulding or 3D-printing.
[0069] The barriers 8 utilize surface entrainment, whereby motile cells 2 tend to swim along a surface, to sort the motile cells. The barriers 8 have curved surfaces 15, 16 with sharp discontinuities to redirect swimming cells along the desired movement orientation.
[0070] Referring to FIGS. 3 and 4, first and second barrier shapes 8.sub.1, 8.sub.2 are shown
[0071] Referring in particular to FIG. 3, the first shape 6.sub.1 generally has an arrowhead-like geometry having two convex segments 15.sub.1,1, 15.sub.1,2 joined at a first edge 17.sub.1,1 and one concave segment 16.sub.1 joined to the convex segments 9.sub.1,1, 9.sub.1,2 via sharp, second and third edges 17.sub.1,2 17.sub.1,3. To sort cells, the barrier 8.sub.1 is oriented such that the side with a single edge 17.sub.1,1 points towards the inlet 5 of the device 8.sub.1. Cells swimming in the desired direction are gently guided along the barrier 8.sub.1 while cells going the wrong direction are turned around by the concave segment 16.sub.1 and reoriented towards the outlet 6.
[0072] The first barrier 8.sub.1 is defined by half the intersection of first and second intersecting virtual circles 18.sub.1, 18.sub.2 having a cut out defined by a third circle 18.sub.3. The convex walls 15.sub.1,1, 15.sub.1,2 are defined by intercepted arcs of the first and second overlapping virtual circles 18.sub.1, 18.sub.2 each having a first radius r.sub.1. The concave wall 16.sub.1 is defined by the intercepted arc of a third virtual circle 18.sub.3 having a second radius r.sub.2. In this case, r.sub.2<r.sub.1. The first barrier 8.sub.1, has a width w.sub.1 of between 10 and 500 μm and a length l.sub.1 of between 10 and 1,000 μm. In this example, r.sub.1=450 μm, r.sub.2=82 μm, w.sub.1=150 μm and l.sub.1=177 μm.
[0073] Referring in particular to FIG. 4, the second shape 8.sub.2 generally has a crescent moon-like geometry having one convex segment 15.sub.2 and one concave segment 16.sub.2 joined via sharp edges 17.sub.2,1, 17.sub.2,2. The edges 17.sub.2,1, 17.sub.2,2 are orientated orient towards the outlet 6 of the device. Cells which encounter the convex side are simply guided along, while cells which encounter the concave side are re-oriented towards the outlet.
[0074] The second barrier 8.sub.2 is defined by third and fourth overlapping virtual circles 18.sub.3, 18.sub.4. The convex and concave walls 15.sub.2, 16.sub.2 are defined by fourth and fifth arcs of the third and fourth circles 18.sub.3, 18.sub.4 having a third radius r.sub.3 and a fourth radius r.sub.4 respectively. The first barrier 8.sub.1, has a width w.sub.2 of between 10 and 500 μm and a length l.sub.2 of between 10 and 1,000 μm. In this example, r.sub.3=125 μm, r.sub.4=137 μm, w.sub.2=250 μm and the two circles are offset by 39 μm.
[0075] Referring to FIG. 5, the chamber 4 takes the form of a channel which is generally linear, running along a path (which may be straight, curved or include bends) between the inlet 5 and outlet 6. The channel 4 has a wall 19 having repeated barbs 20, in this case having a shark-fin-like shape. A rectangular array 7 of barriers 8 of the first shape 8.sub.1 is used. A different type of array, e.g. hexagonal, and/or a different barrier shape, e.g. the second shape 8.sub.2, can be used. The channel 4 may have a different shape.
[0076] Referring to FIG. 6, the chamber 4 may be radial (or “circular”) whereby the motile cells are introduced around a peripheral, annular inlet 5 and swim inwardly towards a central outlet 6. The chamber 4 is defined by a generally circular wall 21 having scallops 22. A radial array 7 of barriers 8 of the second shape 8.sub.2 is used. A different type of array, e.g. hexagonal, and/or a different barrier shape, e.g. the first shape 8.sub.1, can be used.
[0077] Referring again to FIG. 3, the first shape 6.sub.1 of barrier generally has an arrowhead-like geometry having a first face 13 which comprises two convex walls 15.sub.1,1, 15.sub.1,2. However, these walls can have other, different shapes.
[0078] Referring FIGS. 7a to 7b, the first face 13 may have straight walls 15.sub.3,1, 15.sub.3,2 or even concave wall 15.sub.4,1, 15.sub.3,2 meeting the concave wall 16.sub.3, 16.sub.4 at acute edges 17.sub.3,2, 17.sub.3,3, 17.sub.4,2, 17.sub.4,3. For example, the barb may be a half arrowhead, half crescent or half a modified arrowhead (the full modified arrowhead having two concave walls)
[0079] Referring to FIG. 8, if there are two or more walls in the first face 13, the walls 15.sub.5,1, 15.sub.5,2 need not have the same shape. For example, one wall 15.sub.5,1 may be straight and the other wall 15.sub.5,2 may be convex. Thus, the edges 17.sub.5,2, 17.sub.5,3 may have different sharpnesses.
[0080] Referring to FIGS. 9a to 9c, the walls 19.sub.1, 19.sub.2, 19.sub.3 of the chamber 4, can have different shapes of barbs 20.sub.1, 20.sub.2, 20.sub.3 which may be based on the shapes of the barriers herein described.
[0081] Referring to FIGS. 10a and 10b, photographs of a device 1 similar to that shown in FIG. 6 and photographs of barriers 8.sub.1 as shown in FIG. 3 used in these devices are shown. FIG. 10a illustrates a device 1 having inlets 5 with a diameter d.sub.i of 3 mm, and an outlet 6 having a diameter d.sub.0 of 5 mm. This device 1 is also referred to as a 5*3, or 5*3 mm device. The diameter d of the device 1 is between 2.5 cm and 3.5 cm. The 5*3 device 1 includes barriers 8.sub.1, as illustrated in FIG. 3, spaced at a pitch of greater than 100 μm, however, the barriers may have a larger or smaller pitch depending on the sample used. A higher magnification photograph of the barriers 8.sub.1 in the device 1 is shown in the lower part of FIGS. 10a and 10b. A device with these arrowhead-like barriers 8.sub.1 may also be referred to as “Ratchet 1”, or more simply “R.sub.1”. The channel 4 of the 3*5 device 1 is less than 100 μm. FIG. 10b illustrates a device 1 having inlets 5 with a diameter d.sub.i of 5 mm, and an outlet 6 having a diameter d.sub.0 also of 5 mm. This device 1 is also referred to as a 5*5, or 5*5 mm device. The diameter d of the device 1 is 3.5 cm. The barrier 8 structures, pitch and channel 4 are the same in the 3*5 device and the 5*5 device.
[0082] The 5*3 device can yield better results as fewer undesirable cells and other debris are removed at the extraction stage, however, it can be more difficult to extract a sample from the 5*3 device than from the 5*5 device. The 5*5 device can yield a lower quality sample than the 5*5 device, but the sample can be easier to extract from the device.
[0083] The inlet 5 diameter d.sub.i and the outlet diameter d.sub.0 may be between 1 and 8 mm. The diameter d of the device may be between 1 and 10 cm. The sizes of the inlets and outlets can be optimized for the quality and/or type of each sample.
[0084] Referring also to FIG. 3, the barrier 8.sub.1 in the devices in FIGS. 10a and and 10b may have a width w.sub.1 of 125 μm and length l.sub.i of 175 μm.
[0085] The 3*5 device 1 was used to isolate high quality spermatozoa in semen with normal quality spermatozoa using semen samples of 60 μl, and the 5*5 device was used to isolate high quality spermatozoa in semen with normal and poor quality spermatozoa using semen samples of 100 μl.
[0086] Referring to FIGS. 11 and 12, normal and poor quality sperm semen samples are collected and inserted into the device 1 to isolate higher quality sperm.
[0087] Sperm were recovered from 14 ejaculates. Ejaculates were divided into normal and abnormal groups based on sperm progressive motility (PR) by World Health Organisation criteria. Sperm concentration, progressive motility, total motility, morphology and DNA fragmentation (using Terminal deoxynucleotidyl transferase TdT dUTP Nick End Labeling (TUNEL) assay) were measured in the sperm selected by all methods and in the semen sample.
[0088] Referring specifically to FIG. 11, steps are illustrated for the method of isolating high quality spermatozoa in normal sperm quality samples using the device 1. In step S1, the sample is purged using a buffer, for example, Earle's Balanced Salt solution (1×EBSS). Other suitable buffers may be used and the type and concentration will depend on the sample and desired outcome of the process. In step S2, the sample is flushed with a buffer to reduce the change of spermatozoa sticking to each other, for example, a 0.2% bovine serum albumin (BSA) in 1×EBSS. However, other suitable buffers which have the effect of reducing the probability of spermatozoa sticking to each other may be used. In step S3, if there is excess fluid in the sample at this stage, this fluid may be removed from the sample before loading into the device. In step S4, the sample and/or the device is warmed on a 37° C. hotplate. In step S5, the semen sample is divided into four equal parts and loaded into the four inlets 5 of the device 1, however, it may be that only some inlets 5 have a portion of the sample loaded into them. In step S6, the device 1 containing the sample is incubated for 5 minutes at 37° C. and sperm migration is observed either manually using a microscope or automatically using a microscope and video camera to confirm the sample has been loaded correctly. Other methods of confirming that the sample has been loaded correctly may be used, for example, via an optical or movement sensor at the beginning of step S7. In step S7, the device containing the sample is incubated for 60 minutes. In step S8, the sperm is observed either manually or automatically after incubation. In step S9, the sperm is extracted from the device 1 through the outlet 6 using a pipette. The volume extracted from the outlet 6 may be between 35 and 60 μl.
[0089] Referring specifically to FIG. 12, steps are illustrated for the method of isolating high quality spermatozoa in poor sperm quality samples using the device 1. For the poor quality sperm samples, steps S1 to S6 are the same as for the normal quality sperm samples. In step S7, the device containing the sample is incubated for 120 minutes. Steps S8 and S9 are the same as for the normal quality sperm samples. The volume extracted from the outlet 6 may be between 40 and 75 μl.
[0090] Alternative processes may be used in a working device. The steps in the processes outlined above may be modified or removed, for example, step S3, may be removed altogether. Additional steps may also be added, for example, additional washing or purging of a sample and/or device with additional buffers. The timings, temperatures and volumes of sample associated with steps in the processes may be optimized for particular samples, species, or aim of the process, for example the incubation time may be longer or shorter in step S7, depending on the sample type, size, and speed of sperm. The volumes extracted from the device at the end of the process may be higher or lower depending on the types and volumes of samples used and the types and volumes of buffers added to the sample. In a high throughput system, step S6 may also be removed, or combined with step S8, where the observation of the sample is performed at the beginning of step S8.
[0091] Referring to FIG. 13, a table indicates whether a process of a semen sample was successful (indicated by a tick) or not (indicated by a cross). Samples from ten individuals (N1 to N10) with normal quality sperm and six individuals (A1 to A6) with poor quality sperm underwent five methods for isolating high quality sperm. These methods were density gradient centrifugation (DGC), swim up (SU), a method using a 5*3 device and a 5*5 device both with barriers 8.sub.1 as illustrated in FIGS. 2 and 3 (also labelled “R1”), and a method using a device with barriers 8.sub.2 illustrated in FIG. 4 (labelled “R2”). As can be seen from the table, nine samples of normal quality sperm were successfully processed using the 5*3 device 1 and five samples of poor quality sperm were successfully processed using the 5*5 device 1. A total of 4 samples form both normal and poor quality groups were successfully processed using the R2 method.
[0092] Referring to FIGS. 14 to 16, the results from analyses of the samples are presented in tables. The results show the concentration, the percentage progressive motility (% PR), the percentage motility (% Motile), extracted volume, the extracted concentration, percent of sperm showing normal and abnormal morphology (based on World Health Organisation classifications), and the percent of sperm intact or fragmented based on the TUNEL method.
[0093] Referring in particular to FIG. 16, the table detailing the results for the poor quality sperm also indicates whether the sperm has a low motility condition classified as Astheno.
[0094] Referring to FIGS. 17 and 18, the results of the methodologies presented in FIGS. 13 to 16 are presented in summary form.
[0095] Referring to FIG. 19, a bar chart illustrates the percentage progressive motility and percentage motility±the standard error of the mean (±SEM) for the results for normal quality sperm from the methods in the table shown in FIG. 13 and also unprocessed semen. The Figure also contains the results from statistical analysis showing that using a One-way ANOVA with LSD (one-way analysis of variance with least significant difference), the R1 5*3 device yields a sample with significantly higher motility and progressive motility than a raw sample, a sample having undergone density gradient centrifugation and a sample having undergone the swim up method.
[0096] Referring to FIG. 20, a bar chart illustrates the percentage progressive motility and percentage motility±the standard error of the mean (±SEM) for the results for poor quality sperm from three of the methods in the table shown in FIG. 13 and unprocessed semen, specifically density gradient centrifugation and the method using the R1 5*5 device. The One-way ANOVA with LSD also shows a significant difference in percentage progressive motility and motility between the sample extracted from the R1 5*5 device and both the unprocessed semen and the semen having undergone density gradient centrifugation.
[0097] Referring to FIG. 21, a bar chart illustrates the percentage normal morphology±the standard error of the mean for the results for normal quality sperm from the methods in the table shown in FIG. 13 and also unprocessed semen. A One-way ANOVA with LSD also shows a significant difference in percentage normal morphology of those samples extracted from the R5*3 device and unprocessed semen.
[0098] Referring to FIG. 22, a bar chart illustrates the percentage normal morphology±the standard error of the mean for the results for poor quality sperm having undergone density gradient centrifugation and processing using the R1 5*5 device and also unprocessed semen.
[0099] Referring to FIG. 23, a bar chart illustrates the percentage sperm fragmentation±the standard error of the mean for the results for normal quality sperm from the methods in the table shown in FIG. 13 and also unprocessed semen. A One-way ANOVA with LSD also shows a significant difference in percentage fragmentation of those samples extracted from the R1 5*3 and R1 5*5 devices compared to the other methods and unprocessed semen.
[0100] Referring to FIG. 24, a bar chart illustrates the percentage sperm fragmentation±the standard error of the mean for the results for poor quality sperm having undergone density gradient centrifugation and processing using the R1 5*5 device and also unprocessed semen. A One-way ANOVA with LSD also shows a significant difference in percentage fragmentation of those samples extracted from the R1 5*5 device compared to samples having undergone density gradient centrifugation and unprocessed semen.
[0101] Referring to FIG. 25, photographs of fragmented semen at 200× magnification are shown for unprocessed semen, and samples having undergone density gradient centrifugation, swim up and processing using the R1 5*3 device. Fragments are stained to appear light blue and labelled “LB”.
[0102] Referring to FIG. 26, photographs of fragmented semen at 600× magnification are shown for unprocessed semen, and samples having undergone density gradient centrifugation, swim up and processing using the R2 5*3 device. Fragments are stained green (indicated here with a “G”), partially fragmented sperm are stained orange (indicated here with an “O”) and unfragmented and undamaged sperm are stained red, (indicated here with an “R”).
[0103] Modifications
[0104] It will be appreciated that various modifications may be made to the embodiments hereinbefore described. Such modifications may involve equivalent and other features which are already known in the design, manufacture and use of motile cell sorting devices and component parts thereof and which may be used instead of or in addition to features already described herein. Features of one embodiment may be replaced or supplemented by features of another embodiment.
[0105] Concave or convex surfaces need not be defined by arcs of circle. For example, a surface may be defined by an arc of an ellipse, a hyperbola or other suitable curve. The curvature may vary along the surface.
[0106] Although claims have been formulated in this application to particular combinations of features, it should be understood that the scope of the disclosure of the present invention also includes any novel features or any novel combination of features disclosed herein either explicitly or implicitly or any generalization thereof, whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention. The applicants hereby give notice that new claims may be formulated to such features and/or combinations of such features during the prosecution of the present application or of any further application derived therefrom.
