Detailed Action
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 2 and 17 are cancelled.
Claims 1, 3-16, and 18-26 are pending and under examination on the merits.
Withdrawn Claim Objections/Rejections
The objection to claim 1 is/are withdrawn in light of the corrective claim amendments dated 11/11/2025. The rejection of claim 1 under 35 USC §112(b) is withdrawn in light of the corrective claim amendments dated 11/11/2025. The rejections of the claims for double patenting over copending Application No. 17/295,612 (reference A) are withdrawn in light of the acceptance (posted 11/11/2025) of the terminal disclaimer filed 11/11/2025.
Claim Interpretation
The mere recitation of completion of samples as recited in claim 8 does not immediately invoke a clearly defined step or result. In the interest of advancing prosecution, the Examiner interprets this recitation to be met where the conditional steps recited in any one of paragraphs 0027, 0028, 00109, or 00110 are met, given that these paragraphs are the only definition provides throughout the specification as to what could be meant.
Applicant discloses that the recited lin1 (lineage 1 cocktail) is conventional, but fails to further define the makeup of said antibody cocktail. The recitation of lin1 is being interpreted to comprise antibodies against CD3, CD14, CD16, CD19, CD20, and CD56, as is consistent with the state of the art (see for example US 20110225661 A1 at paragraph 0030 and Bioscience (obtained from: https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/clinical-discovery-research/multicolor-cocktails-and-kits-ruo-gmp/anti-human-lineage-cocktail-1-lin-1-cd3-cd14-cd16-cd19-cd20-cd56.340546?tab=product_details)).
Maintained-Claim Objections and Rejections
Claim Objections
Claim 19 is objected to because of the following informalities: The headings within claim 19 (“(I) Isolation of Human Peripheral Blood Mononuclear Cells from Peripheral Blood” and “(II) Preparation of flow cytometric samples”) are improper within the claim (see for example MPEP §608.01(m)).
Claim 19 is further objected to because of the following informalities: The headings within claim 19 (“(10) sample staining” and “(11) sample washing and solution adding”) are improper within the claim (see for example MPEP §608.01(m)).
Appropriate correction is required.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or no obviousness.
Claim(s) 1, 3-6, 8-15, 18, and 21-24, is/are rejected under 35 U.S.C. 103 as being unpatentable over CN 107063982 A, herein after referred to as ‘CWNU’, (as cited on the 05/20/2021 IDS)’ in view of Lutie, W. (What Really is a Buffy Coat?, BIOVT, obtained from: https://bioivt.com/blogs/really-buffy-coat (07/03/2018), Zhou et al. (Establishment and Evaluation of Flow Cytometry Method for Isolation of T Lymphocytes from Peripheral Blood Mononuclear Cells, Progress in Modern Biomedicine, Vol. 17, No. 21, 31 July 2017 (2017-07-31)), Beckman Coulter (Instructions for Use, Citole Flow Cytometer, obtained from: https://www.pedsresearch.org/uploads/blog/doc/Cytoflex-Manual.pdf (2015)), De Boever et al (Flow cytometric differentiation of avian leukocytes and analysis of their intracellular cytokine expression, https://doi.org/10.1080/03079450903473574 (2010)), Sindhi (US 20060275752 A1), Protocols Online (Phosphate buffered saline, last updated 10/3/2016, obtained from: https://www.protocolsonline.com/recipes/phosphate-buffered-saline-pbs/), Maecker et al (Selecting for Reagents for Multicolor Flow Cytometry, Hot Lines Platinum Edition (fall 2006), obtained from https://pedsresearch.org/_files/Selecting_Multicolor_Reagents_Technote.pdf), Bio-Rad (Sample Preparation Protocol, obtained from: https://web.archive.org/web/20170721082805/https://www.bio-rad-antibodies.com/flow-cytometry-sample-preparation.html; as presented in the Office Action dated 09/06/2024), and Wojno et al (Isolation and Identification of Innate Lymphoid Cells (ILCs) for Immunotoxicity Testing. In: DeWitt, J., Rockwell, C., Bowman, C. (eds) Immunotoxicity Testing. Methods in Molecular Biology, vol 1803. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8549-4_21, (2018); as presented in the Office Action dated 09/06/2024).
CWNU discloses a flow cytometry method for detecting chicken peripheral blood T lymphocyte subsets, comprising the following steps:
CWNU teaches collecting chicken peripheral venous blood, preparing anticoagulant blood, and treating with lymphocyte separation solution to obtain peripheral blood leukocytes (note that the rotating speed of centrifugal separation is 2500 r/min, the liquid is divided into three layers after centrifugation for 15 min, and the middle milky white flocculent liquid layer is a peripheral blood leukocyte layer; see for example, page 3/5 of the English translated description); centrifugally washing the peripheral blood leukocytes with a PBS solution to prepare a single-cell suspension (see for example, page 3/5).
CWNU teaches the steps for obtaining peripheral blood leukocytes by treating with the lymphocyte separation solution are as follows: in a flow tube, adding the lymphocyte separation solution (presumed to be PBS) and the anticoagulant blood in a volume ratio of 1:1 (held to read on an equal volume as recited in claim 1 steps c 1-3 after the wherein clause), to ensure a clear boundary between the anticoagulant blood and the lymphocyte separation solution; obtaining the peripheral blood leukocytes after centrifugation at a speed of 2500 r/min (rpm) and with the time of 15 min (see for example, page 3/5 of the English translated description). Note that, where cells from the buffy coat/PBMCs are desired, for example, lymphocytes, methods of obtaining these cells from whole blood by centrifugation are known in the art (see for example Lutie, W., in its entirety, and, for greater detail see further Zhou et al. at pages 4016-4018, especially section 1.4)).
Teaching at least claim 1, step a and claim 3 : CWNU teaches the preparation of the single cell suspension comprising steps of: washing the peripheral blood leukocytes with pre-cooled PBS solution, then resuspending, adjusting to a single cell suspension with a cell concentration of lx106-lx107 cells/mL (wherein CWNU teaches that, if the concentration of the single cell suspension is too high, the staining is insufficient; if the concentration is too low, the cell flow rate is low during on-machine detection, and the accuracy of the detection result is affected), and storing at 4 °C for later use. Note that CWNU teaches that the rotation speed of the centrifugal washing is 600-1000 r/min, and the time is 5 min (see for example, pages 3/5-4/5). CWNU further teaches sucking the single cell suspension, adding anti-chicken CD3, CD4 and CD8 monoclonal antibodies respectively, mixing well by vortex, and staining in the shade (without light) at 4°C (see for example, pages 2/5-4/5);
Teaching at least claim 1, step b and claims 4-6: CWNU and Lutie, W. do not appear to teach the recited limitations of claim 1, step B, but Beckman Coulter teaches that part of using the flow cytometer machine includes starting the machine, priming and warming up the machine (held to read upon the limitation of preheating a flow cytometer system as recited in step b of step 1), and running the machine at a flow speed of 10µl/min (slow) or 30µl/min (medium) or 60µl (fast) with a backflush step (see pages 1-21). Given that the maker of a flow cytometry machine recommends these steps and speeds, one of ordinary skill in the art would have viewed these steps as an obvious matter of choice, which one of ordinary skill in the art would have obviously chosen with a reasonable expectation of success in the aim of optimizing the methods of cell preparation for flow cytometry. The same logic applies to selecting a running speed/flow speed between the art taught ranges of 10-60µl for yielding predictable results which may be further predictably optimized as a means of routine optimization (see MPEP 2141. I). The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. “When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 may bar its patentability. When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions,” (see MPEP 2141. I.). The same logic applies to the recited sample volumes because Beckman Coulter teach that sample size may range from 45-200µl/well per plate (indicating this size would reasonably contain the number of cells desirable for analysis; see Beckman Coulter page 21). Moreover, where the number of cells meets the minimum required for analysis, choosing a number of cells for the sample is an obvious matter of choice, particularly in light of the teachings of CWNU as discussed herein, where the sample volume would appear to be insignificant in terms of the method and the recited volumes would appear to do no more than yield predictable results.
Teaching at least claim 1, step c 4: CWNU goes on to teach the preparation of the single cell suspension comprising steps of: washing the peripheral blood leukocytes with pre-cooled PBS solution, then resuspending, adjusting to a single cell suspension with a cell concentration of lx106-lx107 cells/mL (which one of ordinary skill in the art would have found obvious to modify in light of BioRad, which teaches that single cells must be suspended at a density of 105–107 cells/ml to keep the narrow bores of the flow cytometer and its tubing from clogging up; the concentration also influences the rate of flow sorting, which typically progresses at 2,000–20,000 cells/second; higher sort speeds can result in lower yield or recovery; see entirety of the 1 page reference), and storing at 4 °C for later use (see for example, pages 2/5-4/5). Note that CWNU teaches that the rotation speed of the centrifugal washing is 600-1000 r/min, and the time is 5 min (see for example, pages 3/5-4/5), note that Wojno et al, teaching protocols for preparing lymphoid cells for flow cytometry (FACs), recommends and teaches using a centrifugation at 4 °C for 10 min at 1500 rpm (480 × g) (see for example, sections 3.1, 3.2, and 3.3). Thus, one of ordinary skill in the art would have found it obvious to look to Wojno et al’s parameters (buffers and centrifugation times, temperatures, and speeds) in order to optimize the method of CWNU when looking to perfect a method of preparation, said optimization and perfection being the motive to combine. The parameters are matters of choice yielding predictable results with a reasonable expectation of success and are thereby obvious absent some showing of criticality or surprising result (see the MPEP citation below for further detail);
Teaching at least claim 1, step c 5: CWNU further teaches sucking the single cell suspension, adding anti-chicken CD3, CD4 and CD8 monoclonal antibodies respectively, mixing well by vortex, and staining in the shade (without light) at 4°C where CWNU teaches that anti-CD3-SPRD (SBA, USA), CD4-FITC (SBA-USA) and CD8A-PE (SBA, USA) antibodies were used (see for example page 4/5 of the English translation) which, while not reading upon the recited combination of CD3-FTIC, CD4PerCP, and CD8PerCP does make obvious the use of antibodies against CD3, CD4, and CD8 labeled with known and compatible labels for flow cytometry [noting that Maecker et al teach that FITC and PerCP are compatible labels for flow cytometric analysis; see for example Table 1 and its caption as well as the article title] (note that CWNU does not teach the recited antibodies, however, Sindhi teaches a multiparametric method of assessing a patient’s immune response though flow cytometric analysis of human lymphocytes which antibodies selected from the group consisting of CD4, CD3, CD8, and CD123 (see for example claims 1, 3, 5, 21, and 24; note that the antibodies may be labeled (see paragraph 0015, for example));
Teaching at least claim 1, step c 6-7: CWNU goes on to teach, after staining, washing with the pre-cooled PBS solution, resuspending cells, and detecting with a flow cytometer (while it is not explicitly stated, given that this step uses the pre-cooled PBS and that all other steps with the pre-cooled PBS have occurred at 4°C, it presumed this step, given the pre-cooled PBS, also occurs at 4°C); analyzing detection results, and obtaining the ratio of the chicken peripheral blood T lymphocyte subsets. The flow cytometer is used for detection (implicitly disclosing the steps of adding the sample to be tested into the sample tube and then detecting), comprising adjusting the voltage of a side-scattered light channel to separate the lymphocyte population from the surrounding cells; the current gain of a forward scattered light channel is adjusted to separate the cell clusters from the cell debris; antibodies with identical species source, subtype, and fluorescently labeled primary antibody with the corresponding surface marker of the antibody are selected,
(see for example, CWNU’s abstract, claims 1-9, paragraphs 5-28). Note that Wojno et al teach a protocol for flow cytometric analysis of innate lymphoid cells using a buffer comprising PBS containing 2% FBS, 2 mM EDTA, and 0.1% sodium azide (called the FACs and staining buffer) (see for example, sections 2.3 and 3.3) which is nearly identical to the MACS buffer (PBS (pH 7.2) containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA) (see for example, section 2.2). While Wojno et al are silent as to the pH of the FACs buffer, it is assumed to have a PH near identical, due to nearly identical compositions, to the MACs buffer presumed to have a pH of 7.2 absent evidence to the contrary and the PBS amount is viewed to read on the claimed amount of .005-.05M. Thus, the FACs buffer recommended by Wojno is viewed to make obvious the buffer described by instant claim 2. Moreover, Wojno et al recommends and teaches using a centrifugation at 4 °C for 10 min at 1500 rpm (480 × g) (reading at least on instant claims 5-7 and 16) (see for example, sections 3.1, 3.2, and 3.3 of Wojno et al). Because Wojno et al also teach protocols for preparing lymphoid cells for flow cytometry (FACs), one of ordinary skill in the art would have found it obvious to look to Wojno et al’s parameters (buffers and centrifugation times, temperatures, and speeds) in order to optimize the method of CWNU when looking to perfect a method of preparation, said optimization and perfection being the motive to combine. Note that the above teachings are further held to teach and make obvious the variants of claim 1 recited by instant claims 9 and 11 (where 100µl is held to be an obvious amount because it yields predictable results being compatible with fitting in the tube for flow cytometry), for the reasons noted above.
Moreover, claim 1 is, essentially, a product by process claim in which the process of preparing the cells for flow cytometric detection carries little patentable weight. It is only the product, which is anticipated by the prior art and not the process by which the product was made. This is because the final product (population of stained lymphocytes (buffy coat cells)) is not distinguished by any particular features or characteristics resulting from the process by which it was made. As such, the limitations of the claimed process preparing the cells are met by any process of preparing a population of lymphocytes/buffy coat cells for flow cytometric detection in the prior art. Patentability of a product-by-process claim is determined by the novelty and nonobviousness of the claimed product itself without consideration of the process for making it which are encompassed by the claimed process of preparing said cells of claim 1, which are thereby obvious over the cited prior art as it appears in this rejection.
Moreover, note that the teachings cited above are held to apply to and make obvious the resulting method for human ex vivo immune cell flow cytometry because De Boever et al teach that flow cytometry is a preferred method to phenotype ex vivo derived individual leukocytes from various species wherein the key difference in methods analyzing human versus avian cells would be use of appropriate biomarkers for analysis (see for example the introduction). Therefore, one of ordinary skill in the art would have found it obvious, as of the filing date, to adapt the method of CWNU as modified by the combination of references cited, above, for preparing and analyzing human immune cells with a reasonable expectation of success whereupon minor variations (such as the biomarkers or antibodies used would be obviously swapped for human-compatible biological equivalents (such as swapping a mouse-anti-chicken antibody for antibodies responsive to human biological matter (see MPEP §2144.06(II)).
Wojno et al, while believed to teach the recited PBS solution, is not explicit in teaching the concentration of the PBS.
However, ProtocolsOnline teaches two formulations of PBS: 10X and 1X (note that one skilled in the art would know that 1X PBS is 0.01M PBS), which is taught to have a pH of 7.4 once diluted to a 1X (0.01M) concentration where PBS is known to be, at times, combined with EDTA (see pages 1-3).
It would have been prima facie obvious to the person of ordinary skill in the art to arrive at the claimed invention from the disclosures of the combined references.
The MPEP provides that:
“The Supreme Court in KSR reaffirmed the familiar framework for determining obviousness as set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), but stated that the Federal Circuit had erred by applying the teaching-suggestion-motivation (TSM) test in an overly rigid and formalistic way. KSR, 550 U.S. at 404, 82 USPQ2d at 1391. Specifically, the Supreme Court stated that the Federal Circuit had erred in four ways: (1) “by holding that courts and patent examiners should look only to the problem the patentee was trying to solve ” (Id. at 420, 82 USPQ2d at 1397); (2) by assuming “that a person of ordinary skill attempting to solve a problem will be led only to those elements of prior art designed to solve the same problem” (Id.); (3) by concluding “that a patent claim cannot be proved obvious merely by showing that the combination of elements was ‘obvious to try’” (Id. at 421, USPQ2d at 1397); and (4) by overemphasizing “the risk of courts and patent examiners falling prey to hindsight bias” and as a result applying “[r]igid preventative rules that deny factfinders recourse to common sense” (Id.). See also Novartis Pharms. Corp. v. West-Ward Pharms. Int'l Ltd., 923 F.3d 1051, 1059, 2019 USPQ2d 171676 (Fed. Cir. 2019); Apple Inc. v. Samsung Elecs. Co., 839 F.3d 1034, 1047-48, 120 USPQ2d 1400, 1410 (Fed. Cir. 2016); and Aventis Pharma S.A. v. Hospira, Inc., 675 F.3d 1324, 1332, 102 USPQ2d 1445, 1449 (Fed. Cir. 2012)… Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.”Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id. at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.” Id. (2) “In Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., . . . [t]he two [pre-existing elements] in combination did no more than they would in separate, sequential operation.” Id. at 416-17, 82 USPQ2d at 1395. (3) “[I]n Sakraida v. AG Pro, Inc., the Court derived . . . the conclusion that when a patent simply arranges old elements with each performing the same function it had been known to perform and yields no more than one would expect from such an arrangement, the combination is obvious.” Id. at 417, 82 USPQ2d at 1395-96 (Internal quotations omitted.). The principles underlining these cases are instructive when the question is whether a patent application claiming the combination of elements of prior art would have been obvious. The Supreme Court further stated that:
When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 likely bars its patentability. For the same reason, if a technique has been used to improve one device, and a person of ordinary skill in the art would recognize that it would improve similar devices in the same way, using the technique is obvious unless its actual application is beyond his or her skill. Id. at 417, 82 USPQ2d at 1396.
When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions.” Id,”
(see MPEP §2141(I)).
The artisan would have been motivated to make and use the invention as claimed because De Boever et al teach that flow cytometric cell separation/analysis/labeling is common across/for a variety of species where the primary difference would be selection/adjustment for species-appropriate biomarkers. It is acknowledged that chicken/avian red blood cells (RBCs), unlike mammalian RBCs, have nuclei. However, the methods as presently drafted do not comprise a step which would unpredictably rely upon nucleation, or lack thereof, of the RBCs in the blood sample. The steps, between the method of CWNU and the instant method show a very high degree of similarity and the reagents claimed, being well known in the art, appear to be employed for their art-known functions in both the method of CWNU and the instant method. Therefore, adaptation of the method of CWNU as discussed above as guided by Luty, W. Zhou et al, BioRad, Wojno et al, De Boever et al, Sindhi, Maecker et al, and ProtocolsOnline references, is deemed prima facie obvious and is reasonably expected to predictably function for preparation of human ex vivo cells. The artisan would have had a reasonable expectation of success based on the cumulative disclosures of these prior art references.
Further regarding claim 4, Beckman Coulter, as discussed above, teaches that the minimum sample volume is 45µl and the maximum sample volume is 200µl (see page 21). Therefore, any sample volume within the range of 45-200µl would have been an obvious matter of choice yielding no more than predictable results, absent evidence to the contrary (see MPEP 2141. I).
Regarding claim 8, it is noted that the only difference between claim 1 and claim 8 is that the cell concertation obtained in step c 4 of claim 1 (step 4 of claim 8, depending from claim 1) is narrowed. As discussed above, CWNU teaches the method comprising adjusting to a single cell suspension with a cell concentration of lx106-lx107 cells/mL, which is made obvious for reasons detailed above.
Regarding, claims 9-10, 12-15, and 18: CWNU teaches, after staining, washing with the pre-cooled PBS solution, resuspending cells, and detecting with a flow cytometer (while it is not explicitly stated, given that this step uses the pre-cooled PBS and that all other steps with the pre-cooled PBS have occurred at 4°C, it presumed this step, given the pre-cooled PBS, also occurs at 4°C); analyzing detection results, and obtaining the ratio of the chicken peripheral blood T lymphocyte subsets. The flow cytometer is used for detection (implicitly disclosing the steps of adding the sample to be tested into the sample tube and then detecting), comprising adjusting the voltage of a side-scattered light channel to separate the lymphocyte population from the surrounding cells; the current gain of a forward scattered light channel is adjusted to separate the cell clusters from the cell debris; antibodies with identical species source, subtype, and fluorescently labeled primary antibody with the corresponding surface marker of the antibody are selected.
(see for example, CWNU’s abstract, claims 1-9 and pages 2/5-4/5). Note that Wojno et al teach a protocol for flow cytometric analysis of innate lymphoid cells using a buffer comprising PBS containing 2% FBS, 2 mM EDTA, and 0.1% sodium azide (called the FACs and staining buffer) which is nearly identical to the MACS buffer (PBS (pH 7.2) containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA) (see for example, sections 2.2-2.3 and 3.3). While Wojno et al are silent as to the pH of the MACs buffer, it is assumed to have a pH near identical, due to nearly identical compositions, to the MACs buffer presumed to have a pH of 7.2 absent evidence to the contrary and the PBS amount is viewed to read on the claimed amount of .005-.05M. Thus, the FACs buffer recommended by Wojno is viewed to make obvious the buffer described by instant claim 9. Moreover, Wojno et al recommends and teaches using a centrifugation at 4 °C for 10 min at 1500 rpm (480 × g) (reading at least on instant claims 12-13, 15, and 18; see for example, sections 3.1, 3.2, and 3.3 of Wojno et al). Because Wojno et al also teach protocols for preparing lymphoid cells for flow cytometry (FACs), one of ordinary skill in the art would have found it obvious to look to Wojno et al’s parameters (buffers and centrifugation times, temperatures, and speeds) in order to optimize the method of CWNU when looking to perfect a method of preparation, said optimization and perfection being the motive to combine. Here, the cells would be expected to survive the conditions because Wojno is also looking at lymphocytes and thereby the use of these parameters in any step of the process would carry a reasonable expectation of success such that choosing to use these parameters at any step would have been an obvious matter of choice in the aim of optimizing the method. Note that the above teachings are further held to teach and make obvious the variants of claim 1 recited by instant claims 8 and 19 (where 100µl is held to be an obvious amount because it yields predictable results being compatible with fitting in the tube for flow cytometry), for the reasons noted above.
Regarding claims 11 and 21-24, as discussed in greater detail above, CWNU teaches preparing anticoagulant blood, and treating with lymphocyte separation solution to obtain peripheral blood leukocytes (note that the rotating speed of centrifugal separation is 2500 r/min, the liquid is divided into three layers after centrifugation for 15 min, and the middle milky white flocculent liquid layer is a peripheral blood leukocyte layer; see page 3/5 of the English translated description; reading on instant claim 21); centrifugally washing the peripheral blood leukocytes with a PBS solution to prepare a single-cell suspension (see for example, pages 1/5 and 3/5 of CWNU). CWNU teaches the steps for obtaining peripheral blood leukocytes by treating with the lymphocyte separation solution are as follows: in a flow tube, adding the lymphocyte separation solution (presumed to be PBS) and the anticoagulant blood in a volume ratio of 1:1 (reading on at least instant claims 11 and 22), to ensure a clear boundary between the anticoagulant blood and the lymphocyte separation solution; obtaining the peripheral blood leukocytes after centrifugation at a speed of 2500 r/min (rpm) and with the time of 15 min (reading on claim 23) (see for example, page 3/5 of the English translated description of CWNU). Note that, where cells from the buffy coat/PBMCs are desired, for example, lymphocytes, methods of obtaining these cells from whole blood by centrifugation are known in the art (see for example Luty, W., in its entirety, and, for greater detail see further Zhou et al. at pages 4016-4018, especially section 1.4)). Note, regarding claim 24, that CWNU teaches that the rotation speed of the centrifugal washing is 600-1000 r/min, and the time is 5 min (see for example, pages 3/5-4/5). Note that Wojno et al, teach protocols for preparing lymphoid cells for flow cytometry (FACs), recommends and teaches using a centrifugation at 4 °C for 10 min at 1500 rpm (480 × g (see for example, sections 3.1, 3.2, and 3.3 of Wojno et al). Thus, one of ordinary skill in the art would have found it obvious to look to Wojno et al’s parameters (buffers and centrifugation times, temperatures, and speeds) in order to optimize the method of CWNU when looking to perfect a method of preparation, said optimization and perfection being the motive to combine, as discussed in greater detail above, and made obvious for reasons discussed above.
Claim(s) 7 and 20 rejected under 35 U.S.C. 103 as being unpatentable over CN 107063982 A, herein after referred to as ‘CWNU,’ Luty, W, Zhou et al, Beckman Coulter, and De Boever et al, BioRad, Wojno et al, Sindhi, Maecker et al, and ProtocolsOnline, as applied to claims 1, 3-6, 8-15, 18, and 21-24 above, in view of Vershoor et al (An Introduction to Automated Flow Cytometry Gating Tools and Their Implementation. Front Immunol. 2015 Jul 27;6:380. doi: 10.3389/fimmu.2015.00380) and CDC (Guidelines for Performance of CD4+ T-Cell Determinations in Persons with Human Immunodeficiency Virus Infection, Centers for Disease Control (CDC), obtained from: https://www.cdc.gov/mmwr/preview/mmwrhtml/00019952.htm (available at least as of 01/14/2017 as evidenced by Wayback Machine).
Regarding claim 7, as discussed above, CWNU teaches the limitations of instant claim 1. CWNU does not explicitly teach gating of 300-20,000 cells in step C of claim 1. However, Vershoor et al teach that:
“One of the most basic principles of FCM analysis is “gating,” which is the sequential identification and refinement of a cellular population of interest using a panel of molecules (also known as markers) that are visualized by fluorescence in a unique emission spectrum. For example, if a researcher is interested in quantifying the proportions of CD4 expressing T helper cells and CD8 expressing cytotoxic T-cells in peripheral blood, he/she may use a combination of antibodies (which specifically recognize the marker of interest) conjugated to unique fluorescent dyes that will accurately identify these cells, while discriminating other cell types that are not of interest. In the example of Figure Figure2,2, the cells of interest that are being selected (gated) express CD45 (a pan-leukocyte marker), CD3 (a marker specific to mainly T-lymphocytes), and CD4, or CD8. Cells that are not of interest and will be “gated out” also express CD45, but uniquely express CD14 (commonly expressed on monocytes), CD15 (commonly expressed on neutrophils), or CD19 (commonly expressed on B-lymphocytes). Thus, according to the fluorescence of dyes conjugated to antibodies recognizing each marker, the researcher will be able to identify T helper cells and cytotoxic T-cells, which may also be labeled CD45+CD14−CD15−CD19−CD3+CD4+CD8− and CD45+CD14−CD15−CD19−CD3+CD4−CD8+, respectively. Additionally, based on the level of fluorescence for a given marker, the researcher can also measure the degree to which that molecule is being expressed by the cell of interest,”
(see the Gating section at pages 5/27-6/27). This indicates that gating is a basic, standard technic which one of ordinary skill in the art would have found obvious to use in a method of flow cytometry (such as the method of claim 1) to optimize the method with a reasonable expectation of success.
Vershoor et al do not explicitly indicate a number of cells to be gated.
However, CDC teaches gating at least 2,500 cells (see CDC in its entirety, especially point H. 2.) where gating at least this number of cells (lymphocytes as taught by CDC) assures with 95% confidence that the result is less than or equal to 2% (standard deviation (SD)) of the “true'' value (binomial sampling), providing statistical security (see for example, section H at page 7/14). Therefore, one of ordinary skill in the art would have further found it obvious as of the filing date and been motivated to gate at least 2,500 lymphocytes for statistical security.
Regarding claim 20, as discussed above, CWNU teaches the limitations of instant claim 1. CWNU does not explicitly teach gating of 300-20,000 cells in step C of claim 1. CDC teaches gating of at least 2,500 cells, but does not provide an upper limit of cells for gating (see for example, section H at page 7/14). Stepping in to further guide one of ordinary skill in the art to a gating of 15,000 cells, BioRad teaches that single cells must be suspended at a density of 105–107 cells/ml to keep the narrow bores of the flow cytometer and its tubing from clogging up. The concentration also influences the rate of flow sorting, which typically progresses at 2,000–20,000 cells/second, wherein higher sort speeds can result in lower yield or recovery (see the 1 page reference BioRad in its entirety). From the art-taught range of 2,500-20,000 cells, choosing 15,000 cells would have been mere routine optimization.
Applicant’s attention is drawn to MPEP 2144.05(I),
“In the case where the claimed ranges “overlap or lie inside ranges disclosed by the prior art” a prima facie case of obviousness exists. In reWertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In reWoodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990) (The prior art taught carbon monoxide concentrations of “about 1-5%” while the claim was limited to “more than 5%.” The court held that “about 1-5%” allowed for concentrations slightly above 5% thus the ranges overlapped.); In re Geisler, 116 F.3d 1465, 1469-71, 43 USPQ2d 1362, 1365-66 (Fed. Cir. 1997) (Claim reciting thickness of a protective layer as falling within a range of “50 to 100 Angstroms” considered prima facie obvious in view of prior art reference teaching that “for suitable protection, the thickness of the protective layer should be not less than about 10 nm [i.e., 100 Angstroms].” The court stated that “by stating that ‘suitable protection’ is provided if the protective layer is ‘about’ 100 Angstroms thick, [the prior art reference] directly teaches the use of a thickness within [applicant’s] claimed range.”)… Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of Americav.Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985)… A range can be disclosed in multiple prior art references instead of in a single prior art reference depending on the specific facts of the case. Iron Grip Barbell Co., Inc. v. USA Sports, Inc., 392 F.3d 1317, 1322, 73 USPQ2d 1225, 1228 (Fed. Cir. 2004).”
Applicant’s attention is further drawn to MPEP 2144.05(II)(A), Routine Optimization - Optimization Within Prior Art Conditions or Through Routine Experimentation:
Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.). For more recent cases applying this principle, see Merck & Co. Inc. v. Biocraft Lab. Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997); Smith v. Nichols, 88 U.S. 112, 118-19 (1874) (a change in form, proportions, or degree “will not sustain a patent”); In re Williams, 36 F.2d 436, 438 (CCPA 1929) (“It is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means, is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions.”). See also KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007) (identifying “the need for caution in granting a patent based on the combination of elements found in the prior art.”).
Although this passage does not specifically point to, for example, compositions of immunoglobulins, this passage points to numerous variables that affect the function of inventions, such as concentration of reagents and pH ranges. Furthermore, this passage indicates that the optimization of such variables is often obvious activity for one of ordinary skill in the art. It is submitted that the claimed number of gated cells, are akin to the variables discussed in the cited MPEP passage, because said concentrations and ranges are optimizable variables that would affect at least the toxicity and/or efficacy and/or stability, i.e., function, of the claimed invention. Given the “normal desire of scientists or artisans to improve upon what is already generally known,” it would have been prima facie obvious to one of ordinary skill in the art to optimize the claimed number of gated cells, because such optimization would produce a more effective invention.
Also, as set forth in MPEP 2144.05(II)(B), There is a Motivation to Optimize Result-Effective Variables:
In In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977), the CCPA held that a particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation, because “obvious to try” is not a valid rationale for an obviousness finding. In KSR International Co. v. Teleflex Inc., 550 U.S. 398 (2007), the Supreme Court held that “obvious to try” was a valid rationale for an obviousness finding, for example, when there is a “design need” or “market demand” and there are a “finite number” of solutions. 550 U.S. at 421 (“The same constricted analysis led the Court of Appeals to conclude, in error, that a patent claim cannot be proved obvious merely by showing that the combination of elements was ‘[o]bvious to try.’ ... When there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense. In that instance the fact that a combination was obvious to try might show that it was obvious under §103.”). Thus, after KSR, the presence of a known result-effective variable would be one, but not the only, motivation for a personal of ordinary skill in the art to experiment to reach another workable product or process.
In the instant case, the claims are drawn to the number of cells to be gated for use in a method of analyzing human immune cells with flow cytometry, and this variable achieves a recognized result, such as enabling analysis without clogging as taught by BioRad. Accordingly the recited concentrations and ranges are result-effective variables encompassed within the art-disclosed ranges that achieve a recognized result, such as flow cytometric analysis, and it is submitted that since one of ordinary skill in the art would have thus been motivated to determine the optimum or workable ranges of said variables, the limitation of gating 15,000 cells as recited would have been obvious to one of ordinary skill in the art at the effective filing date of the invention as a result effective variable for optimization wherein any gated number of cells within the art disclosed ranges would have been obvious to use (see MPEP section 2143).
Further, as part of determining obviousness, it is to be considered that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 may bar its patentability. When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions,” (see MPEP 2141. I.).
In the instant case, the use of any number of cells 2,000-20,000 as disclosed by BioRad, would be deemed to predictably function for flow cytometric analysis given that the prior art is presumed enabled (see MPEP §2121(I)), whereby, the prior art is presumed to function absent a convincing showing to the contrary.
Claim(s) 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over CN 107063982 A, herein after referred to as ‘CWNU,’ in view of Luty, W., Zhou et al, BioRad, Wojno et al, De Boever et al, Sindhi, ProtocolsOnline, and Maecker et al, as applied to claims 1, 3-6, 8-15, 18, and 21-24, above, in further view of R&D systems (Flow Cytometry Staining Overview, obtained from: https://www.rndsystems.com/resources/protocols/flow-cytometry-protocol-staining-membrane-associated-proteins-suspended-cells (available as of 07/10/2017 as evidenced by Wayback machine).
As discussed above, the combination of CWNU, Luty, W., Zhou et al, BioRad, Wojno et al, Sindhi, Maecker et al, ProtocolsOnline, De Boever et al, and Beckman Coulter teach instant claim 8. These references do not appear to explicitly teach that 2ml of PBS is added to wash the unbound antibody from the sample. The references do however teach the use of PBS and wash steps (see for example CWNU and Zhou et al in their entirety). R&D systems teach a washing step using 2 mls of flow cytometry staining buffer followed by centrifugation (see for example, point 3 under the Procedure heading at page 1).
Therefore, one of ordinary skill in the art would have found it obvious to look to the protocol of R&D systems to optimize the method of CWNU as modified by the combined references and would have found it an obvious matter of choice to use 2ml of PBS as the washing fluid to remove unbound antibody, expected to yield no more than predictable results (see MPEP 2141. I). Moreover, claim 8 is, essentially, a product by process claim in which the process of preparing the cells for flow cytometric detection carries little patentable weight. It is only the product, which is anticipated by the prior art and not the process by which the product was made. This is because the final product (population of stained lymphocytes (buffy coat cells)) is not distinguished by any particular features or characteristics resulting from the process by which it was made. As such, the limitations of the claimed process preparing the cells are met by any process of preparing a population of lymphocytes/buffy coat cells for flow cytometric detection in the prior art. Patentability of a product-by-process claim is determined by the novelty and nonobviousness of the claimed product itself without consideration of the process for making it which are encompassed by the claimed process of preparing said cells of claim 8, which are thereby obvious over the cited prior art as it appears in this rejection.
Claim(s) 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over CWNU in view of Luty, W., Zhou et al, Beckman, Wojno et al, BioRad, Sindhi, Maecker et al, ProtocolsOnline, De Boever et al, as applied to claims 1, 3-6, 8-15, 18, and 21-24, above, in further view of Valeri et al (Cryopreservation of human blood products, Transfusion and Apheresis Science, Volume 34, Issue 3, 2006, Pages 271-287, ISSN 1473-0502, https://doi.org/10.1016/j.transci.2005.11.010.
CWNU discloses a flow cytometry method for detecting chicken peripheral blood T lymphocyte subsets, comprising the following steps:
Teaching at least claim 19, steps 1-7: CWNU teaches collecting chicken peripheral venous blood, preparing anticoagulant blood (note that secondary reference Zhou et al teaches collection of human blood and preparation thereof to isolate the buffy coat; further note that the species of whole/peripheral blood used, as between avian and human, would not seem to bear any consequence upon the reasonable expectation of success of the instantly claimed method or the method resulting from the combination of CWNU, Luty, W., Zhou et al, Wojno et al, and Backman Coulter because the same cell types are being examined/prepared; note further still that the volume of sample taken and saved would appear to bear no relevance on the instant method’s reasonable expectation of successful method so long as the number of cell counted is sufficient for the method to proceed), and treating with lymphocyte separation solution to obtain peripheral blood leukocytes (note that the rotating speed of centrifugal separation is 2500 r/min, the liquid is divided into three layers after centrifugation for 15 min, and the middle milky white flocculent liquid layer is a peripheral blood leukocyte layer; see page 3/5 of the English translated description; note further that it is unclear why slowly pipetting to preserve stratification of the layers would impact the method when the next step is centrifugation at art recognized speeds and times where the prior art is silent as to whether stratification between the diluent and separation solution was maintained or not, thereby this step is viewed as an obvious matter of choice yielding no more than predictable results (see MPEP 2141. I) read upon by the teachings of the prior art which would have presumably recognized the importance of this step by mentioning slow pipetting if it were important to the art-taught methods). Note further that Valerie et al teach that storage of blood products at -80 degrees Celsius is standard, well known, and successful for plasma up to 14 years where storage may be to enable future use the plasma for resuspension of other products or for other future experimental/therapeutic uses (see for example, Valeri at the final paragraph of the Introduction). Note further that the amount of plasma saved/stored would seem to be a matter of choice which would have no impact upon the method, but which would be based upon the desire (as guided by intended use) of the collector/one of ordinary skill in the art as limited by availability of plasma and capacity for storage. Centrifugally washing the peripheral blood leukocytes with a PBS solution to prepare a single-cell suspension is made obvious by the prior art, as discussed above (see for example, pages 1/5 and 3/5 of CWNU). CWNU teaches the steps for obtaining peripheral blood leukocytes by treating with the lymphocyte separation solution are as follows: in a flow tube, adding the lymphocyte separation solution (presumed to be PBS) and the anticoagulant blood in a volume ratio of 1:1, (note that the size of the tube used would seem to be an obvious matter of choice yielding no more than predictable results (see MPEP 2141. I) and dependent upon the machine used and the experimental design needs) to ensure a clear boundary between the anticoagulant blood and the lymphocyte separation solution; obtaining the peripheral blood leukocytes after centrifugation at a speed of 2500 r/min (rpm) and with the time of 15 min (see for example, pages 2/5-4/5). Note that, where cells from the buffy coat/PBMCs are desired, for example, lymphocytes, methods of obtaining these cells from whole blood by centrifugation are known in the art (see for example Luty, W., in its entirety, and, for greater detail see further Zhou et al. at pages 4016-4018, especially section 1.4)). The means for removing the upper plasma layer for removal would seem to bear little consequence upon the instant method so long as the lower layers are preserved as would be the clear goal of centrifugation. The use of pipetting techniques to remove a layer for disposal are well-known in the art (see the multiple removal steps as they occur in Zhou et al and CWNU for example as described in the references, respectively, in their entirety). Therefore, the means of pipetting with a 10ml (standard pipette) or by a 3ml Pasteur pipette would appear to be obvious as a matter of choice for means of removing the upper plasma layer for disposal after centrifugation, slowly so as to avoid mixing of the upper plasma and lower layers stratified by centrifugation.
Teaching at least claim 19, steps 8-11: One of ordinary skill in the art desiring a single cell suspension of buffy coat cells would have found it obvious to pipette the cells into a new tub (where size is a matter of choice depending upon experimental capacity and desire with a 15ml tube being s standard tube known in the art, yielding no more than predictable results; see MPEP 2141. I). It is unclear what significance pipetting by circles would have in the method as claimed apart from enhancing reproducibility by promoting uniformity of the pipetted samples/portions which would seem to merely yield predictable results (see MPEP 2141. I), absent evidence to the contrary. Moreover, claim 19 is, essentially, a product by process claim in which the process of preparing the cells for flow cytometric detection carries little patentable weight. It is only the product, which is anticipated by the prior art and not the process by which the product was made. This is because the final product (population of stained lymphocytes (buffy coat cells)) is not distinguished by any particular features or characteristics resulting from the process by which it was made. As such, the limitations of the claimed process preparing the cells are met by any process of preparing a population of lymphocytes/buffy coat cells for flow cytometric detection in the prior art. Patentability of a product-by-process claim is determined by the novelty and nonobviousness of the claimed product itself without consideration of the process for making it which are encompassed by the claimed process of preparing said cells of claim 19, which are thereby obvious over the cited prior art as it appears in this rejection. CWNU teaches the preparation of the single cell suspension comprising steps of: washing the peripheral blood leukocytes with pre-cooled PBS solution, then resuspending, adjusting to a single cell suspension with a cell concentration of lx106-lx107 cells/mL (wherein CWNU teaches that, if the concentration of the single cell suspension is too high, the staining is insufficient; if the concentration is too low, the cell flow rate is low during on-machine detection, and the accuracy of the detection result is affected), and storing at 4 °C for later use. Note that it is unclear why the volume of sample or PBS taken should matter given the cell count is adjust based on the counted cells thereby volume would seem to be a result dependent variable open to routine optimizations wherein any volume that accommodates the tubes used in the machinery involved would be expected to function, yielding no more than predictable results (see MPEP 2141. I). Note that CWNU teaches that the rotation speed of the centrifugal washing is 600-1000 r/min, and the time is 5 min (see for example, pages 3/5-4/5). Note that Wojno et al, teach protocols for preparing lymphoid cells for flow cytometry (FACs), recommends and teaches using a centrifugation at 4 °C for 10 min at 1500 rpm (480 × g) (see for example, sections 3.1, 3.2, and 3.3 of Wojno et al). Thus, one of ordinary skill in the art would have found it obvious to look to Wojno et al’s parameters (buffers and centrifugation times, temperatures, and speeds) in order to optimize the method of CWNU when looking to perfect a method of preparation, said optimization and perfection being the motive to combine. The parameters are matters of choice yielding predictable results with a reasonable expectation of success and are thereby obvious absent some showing of criticality or surprising result (see the MPEP citation below for further detail). CWNU further teaches sucking the single cell suspension, adding anti-chicken CD3, CD4 and CD8 monoclonal antibodies respectively, mixing well by vortex, and staining in the shade (without light) at 4°C (presumably for 30 minutes; see CWNU’s claim 5). Note that CWNU teaches that after antibody addition and mixing/standing/staining, that the cells are washed (presumably with PBS whereupon centrifugation only yields the predictable step of enhancing the wash to remove unbound antibody prior to testing with the flow cytometer, absent evidence to the contrary, and would have been an obvious matter of choice as of the filing date in order to optimize the method) and testing on/with a flow cytometer;
Beckman Coulter (Instructions for Use, Cytoflex Flow Cytometer, obtained from: https://www.pedsresearch.org/uploads/blog/doc/Cytoflex-Manual.pdf (2015)) teaches that part of using the flow cytometer machine includes starting the machine, priming and warming up the machine, and running the machine at a flow speed of 10µl/min (slow) or 30µl/min (medium) or 60µl (fast) with a backflush step (see pages 1-21). Given that the maker of a flow cytometry machine recommends these steps and speeds, on of ordinary skill in the art would have viewed these steps as an obvious matter of choice, which one of ordinary skill in the art may have obviously chosen with a reasonable expectation of success in the aim of optimizing the methods of cell preparation for flow cytometry. The same logic applies to selecting a running speed/flow speed between the art taught ranges of 10-60µl for yielding predictable results which may be further predictably optimized as a means of routine optimization. The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. “When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 may bar its patentability. When considering obviousness of a combination of known elements, the operative question is thus “whether the improvement is more than the predictable use of prior art elements according to their established functions,” (see MPEP 2141. I.). The same logic applies to the recited sample volumes because Beckman Coulter teach that sample size may range from 45-200µl/well per plate (indicating this size would reasonably contain the number of cells desirable for analysis; see Beckman Coulter page 21). Moreover, where the number of cells meets the minimum required for analysis, choosing a number of cells for the sample is an obvious matter of choice, particularly in light of the teachings of CWNU as discussed herein, where the sample volume would appear to be insignificant in terms of the method and the recited volumes would appear to do no more than yield predictable results.
Note that Zhou et al teach that 10 ml of solution (here it is to be understood that PBS would be encompassed within the physiological saline term used or would be an equivalent to be used with a reasonable expectation of success given the ubiquity of PBS in similar protocols) to a tube for centrifugation prior to draining supernatant and resuspending and adjusting for a desired cell count (see for example, pages 4-5). Given that there is a degree of indefiniteness regarding which step of adding solution/PBS the instant recitation of 10-15 mls of instant claim 19 refers to, the Examiner, in light of the disclosure and the prior art teachings is interpreting this recitation to refer to the first step of the addition, prior to centrifugation in step 11 of claim 19.
CWNU goes on to teach, after staining, washing with the pre-cooled PBS solution, resuspending cells, and detecting with a flow cytometer (while it is not explicitly stated, given that this step uses the pre-cooled PBS and that all other steps with the pre-cooled PBS have occurred at 4°C, it presumed this step, given the pre-cooled PBS, also occurs at 4°C); analyzing detection results, and obtaining the ratio of the chicken peripheral blood T lymphocyte subsets. The flow cytometer is used for detection (implicitly disclosing the steps of adding the sample to be tested into the sample tube and then detecting), comprising adjusting the voltage of a side-scattered light channel to separate the lymphocyte population from the surrounding cells; the current gain of a forward scattered light channel is adjusted to separate the cell clusters from the cell debris; antibodies with identical species source, subtype, and fluorescently labeled primary antibody with the corresponding surface marker of the antibody are selected.
(see CWNU’s abstract, claims 1-9, paragraphs 5-28, and the reference in its entirety). Note that Wojno et al teach a protocol for flow cytometric analysis of innate lymphoid cells using a buffer comprising PBS containing 2% FBS, 2 mM EDTA, and 0.1% sodium azide (called the FACs and staining buffer) which is nearly identical to the MACS buffer (PBS (pH 7.2) containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA). While Wojno et al are silent as to the pH of the MACs buffer, it is assumed to have a PH near identical, due to nearly identical compositions, to the MACs buffer presumed to have a pH of 7.2-7.4 absent evidence to the contrary and the PBS amount is viewed to read on the claimed amount of .005-.05M (see sections 2.2-2.3 and 3.3 of Wojno et al). Thus, the FACs buffer recommended by Wojno is viewed to make obvious the buffer described by instant claim 9. Moreover, Wojno et al recommends and teaches using a centrifugation at 4 °C for 10 min at 1500 rpm (480 × g) (reading at least on instant claims 12-13, 15, and 18) (see for example, sections 3.1, 3.2, and 3.3 of Wojno et al). Because Wojno et al also teach protocols for preparing lymphoid cells for flow cytometry (FACs), one of ordinary skill in the art would have found it obvious to look to Wojno et al’s parameters (buffers and centrifugation times, temperatures, and speeds) in order to optimize the method of CWNU when looking to perfect a method of preparation, said optimization and perfection being the motive to combine. Here, the cells would be expected to survive the conditions because Wojno is also looking at lymphocytes and thereby the use of these parameters in any step of the process would carry a reasonable expectation of success such that choosing to use these parameters at any step would have been an obvious matter of choice in the aim of optimizing the method. Note that the above teachings are further held to teach and make obvious the variants of claim 1 recited by instant claims 8 and 19 (where 100µl is held to be an obvious amount because it yields predictable results being compatible with fitting in the tube for flow cytometry), for the reasons noted above.
Claim(s) 25-26 is/are rejected under 35 U.S.C. 103 as being unpatentable over CWNU in view of Luty, W., Zhou et al, Beckman, Wojno et al, BioRad, Sindhi, Maecker et al, ProtocolsOnline, De Boever et al, and Valeri et al, as applied to claim 19, above, in further view of Mei et al (Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry. J Immunol. 2015 Feb 15;194(4):2022-31. doi: 10.4049/jimmunol.1402661. Epub 2015 Jan 21).
As discussed above, the combined references are held to make obvious instant claim 19. Note that while CWNU does not teach staining occurs under these conditions for 20 minutes, Mei et al teach staining at 4 degrees Celsius for 20 minutes whereupon it would have been an obvious matter of choice to incubate/stain in the dark as taught in CWNU with the motivation to reduce staining time from 30 minutes to 20 minutes in order to promote time efficiency where there is a reasonable expectation of success given the teaching of Mei et al that 20 minutes at 4 degrees Celsius is sufficient for antibody staining (see for example paragraph 2 of the Barcoding and immunophenotyping of PBMC section of Mei et al). The artisan would have had a reasonable expectation of success.
Applicant’s Arguments and Responses:
A. Applicant provides a different translation of CWNU (see exhibit A appended to the 11/18/2025 remarks). Applicant argues that this translation is where CWNU allegedly differentiates chicken and mammalian cells allegedly showing that the method of CWNU is not readily applicable to mammalian cells. Applicant argues that the cited secondary references do not negate their cited teaching of CWNU (the translated paragraph 0063 of Exhibit A) and do not make obvious adaptation from chicken cells to mammalian cells.
Response: First, the source of the Applicant’s allegedly corrected translation has not been established and thus the official WO translation has to be presumed accurate, absent appropriately documented evidence to the contrary. See further the Notice section at page 8 (after the cover page) of Exhibit A as appended to the 11/11/2025 remarks (denoted as page 1 in Exhibit A) noting that is a machine translation which cannot be guaranteed to be accurate such that critical decisions should not be based upon this machine-translation output. Applicant is directed to MPEP §71601(c), which provides that arguments by applicant cannot take the place of evidence, which must be supported by actual proof to be of probative value. Here, there are different mechanical translations, each of presumptively equal validity. Second, even if Applicant’s translation were presumed true and accurate, which is not conceded, the teaching reflected by Applicant’s alleged translation would still not alter the teaching of CWNU so as to teach away from or discredit adaptation of the method of CWNU for application with human ex vivo lymphocytes and would therefore not overcome the rejections of record as presented in this Office Action. Even assuming the Applicant’s translation is accurate, the cited portion only points to variables to be routinely optimized for adaptation from avian to mammalian samples. While CWNU does state that the method taught is for chicken cells, there is nothing to teach away from or discredit adaptation of the method of CWNU for human cell analysis. De Boever et al, as discussed above, teach that flow cytometry is commonly used on cells from all species and that adaptation of flow cytometry for human cells is understood in the art to predominantly involve alteration of lysis steps to account for differences in nucleation of RBCs and adjustment of biomarkers. Note that, the MPEP provides that,
“A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton.”KSR, 550 U.S. at 421, 82 USPQ2d at 1397. “[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle.”Id. at 420, 82 USPQ2d at 1397. Office personnel may also take into account “the inferences and creative steps that a person of ordinary skill in the art would employ.”Id. at 418, 82 USPQ2d at 1396,”
(see MPEP § 2141 (II)(c)).
Where De Boever et al teach that flow cytometry is commonly used for analysis of cells from various species, including mammalian (human) and avian (chicken) species (see for example, paragraph 1 of page 2/19, page 11/19).
The De Boever reference (as discussed in greater detail in the rejections above) is deemed to support that a method of preparing chicken cells would in fact be relevant to and make obvious a method for preparing human cells (where such adaptation would have been routine and obvious in the art). Note that it does not appear that the cited portion of CWNU teaches any reason that the method may not be used for human cells. In fact, the reference states that it should be understood that the invention is not limited to the forms disclosed therein (see final paragraph of page 5/5 of the English translation of the description of CWNU). Applicant argues 4 differences between avian and mammalian samples, focusing heavily on the difference that avian RBCs are nucleated and mammalian RBCs are not. The Examiner notes these differences. However, no argument is made as to how these differences would impact the method of CWNU being adapted as guided by the cited references for use with human ex vivo cells. Applicant has not clearly articulated any technical hurdle which would cast doubt on the teachings of De Boever which support that adaptation of flow cytometric methods is predictable as flow cytometry is commonly used on mammalian and avian species. Thus, the cited secondary references are deemed to supplement and make obvious adaptation of the method of CWNU for human/mammalian cells. Therefore, there is no argument for the Examiner to consider beyond assessing the differences articulated by Applicant, none of which appear to be relied upon for the method of CWNU and which do not overcome the support for such adaptation articulated by De Boever.
Applicant has failed to articulate a substantive, non-obvious difference in the active steps and products used therein which would distinguish the claimed methods from the method made obvious by the prior art combination.
Therefore, this line of argument is deemed unpersuasive and the rejections of record are maintained.
Conclusion
No claim is allowed.
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/Ashley Gao/
Examiner, Art Unit 1678
/GREGORY S EMCH/Supervisory Patent Examiner, Art Unit 1678
Prosecution Insights