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, 10, and 12 are cancelled.
Claims 1, 3, 5-7, 9, 11, 13, and 15-19 are amended.
Claims 1, 3-9, 11, and 13-19 are pending and under examination on the merits.
Withdrawn Objections and Rejections
The objections to the claims are withdrawn as corrected by the 11/05/2025 claim amendments. The rejection of the claims under 35 USC §112(b) are withdrawn as corrected by the 11/05/2025 clarifying claim amendments. The rejections of claim 10 are withdrawn as moot in light of cancellation of the claim by the claim amendments dated 11/05/2025. The rejections of the claims for double patenting over copending Application No. 17/295,655 (reference A) are withdrawn in light of the acceptance (posted 11/05/2025) of the terminal disclaimer filed 11/05/2025.
Claim Interpretation
The recitation of ‘counting’ as recited in claim 11 is being interpreted, consistent with Applicants remarks dated 05/28/2025 (see for example section B) to refer to counting of the cells in the sample (cell-counting) which is so well known in the art, that one of ordinary skill would have understood the recitation to refer to cell-counting by any means known in the art.
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 BDBioscience (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 Rejections
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 nonobviousness.
Claim(s) 1, 3-9, 11, 13-14 and 16 is/are rejected under 35 U.S.C. 103 as being unpatentable over CN 107063982 A, herein after referred to as ‘CWNU’ (as presented in the Office Action dated 09/06/2024), in view of Luty, W. (What Really is a Buffy Coat?, BIOVT, obtained from: https://bioivt.com/blogs/really-buffy-coat (07/03/2018; as presented in the Office Action dated 09/06/2024), 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); as cited on the 05/20/2021 IDS), BioRad (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), 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), 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), ProtocolsOnline (Phosphate buffered saline, last updated 10/3/2016, obtained from: https://www.protocolsonline.com/recipes/phosphate-buffered-saline-pbs/) and 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).
CWNU discloses a flow cytometry method for detecting chicken peripheral blood T lymphocyte subsets, comprising the following steps:
Teaching at least claim 1, steps 1-2, and claim 3: 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 page 3/5 of the English translated description); centrifugally washing the peripheral blood leukocytes with a PBS solution to prepare a single-cell suspension.
Teaching at least claim 1, step 3, and claims 4, 8, and 13-14: 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 (presumably after step 2, such that the blood is a blood cell dilution) in a volume ratio of 1:1 (reading on at least instant claims 4, 8, and 14), 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. 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).
Teaching at least claim 1, step 4, and claim 6: 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), 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, 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). 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 therefore obvious absent some showing of criticality or surprising result (see the MPEP citation below for further detail);
Teaching at least claim 1, step 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, steps 6-7 and claim 9: 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).
CWNU does not appear to explicitly teach the recited PBD-EDTA concentration recited.
However, 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; note that while the concentration of PBS in the buffer of Wojno et al is not specified, it is presumed to fall within the recited .005-.05M given that all other parameters (including the pH) are identical and therefore the buffer of Wojno et al is held to make obvious the instantly used PBS-EDTA buffer of claim 1). 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. Thus, the FACs buffer recommended by Wojno is viewed to be compatible for use in the claimed method(s). Moreover, Wojno et al recommends and teaches using centrifugation at 4 °C for 10 min at 1500 rpm (480 × g) (reading at least on instant claims 5-7 and 16). 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.
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 and reference in its entirety). There is no indication in the cited references that the method of CWNU as modified by the combined references would not function when applied to mammalian/human cells.
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 CWNU and Luty, W. Zhou et al, BioRad, Wojno et al, De Boever et al, Sindhi, Maecker et al, and ProtocolsOnline.
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.
Claim(s) 17 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, Maecker et al, and ProtocolsOnline, as applied to claims 1, 3-9, 11, 13-14 and 16, above, in further view of Dartmouth (Solutions, obtained from: https://web.archive.org/web/20180206072244/https://geiselmed.dartmouth.edu/dartlab/services/flow-cytometry/solutions/ (available as of Feb. 06, 2018, as evidenced by Wayback machine),
Regarding claim 17, as discussed above, CWNU, Luty, Zhou et al, BioRad, Wojno, De Boever, Sindhi, Maecker et al, and ProtocolsOnline teach the methods of claims 1 and 9. However, the combined references do not clearly teach step B of claim 9 wherein the formalin concentration is 1%. Dartmouth remedies this deficiency by teaching that, presumably when flow cytometry cannot be immediately performed, cells stained for surface antigens and then fixed with formaldehyde at 1% (final concentration) should be stored for at least 24 hours (in the dark and at 4°C) and then read on a flow cytometer within 2 weeks (although most cells with most stains will probably last much longer) (see reference in its entirety). Therefore one of ordinary skill in the art would have found the use of 1% formalin as the fixing agent in PBS (a known buffer, and the one used in the primary CWNU reference) to have been obvious as of the filing date with a reasonable expectation of success for storing the samples for at least 2 weeks, if not for much longer for delayed flow cytometric analysis. Note that the remainder of step B of claim 9 merely indicates a basic wash step (wash with PBS, centrifuge and discard supernatant (so as to remove dead cells and formalin) so that the process may proceed for flow cytometric analysis as taught by step A (made obvious by the combined references; with resuspension (so as to be evenly mixed) in PBS-EDTA at 2-8 degrees Celsius) which would have been obvious to one of ordinary skill in the art to revive the sample for analysis according to the method of the combined references after a period of storage.
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 CWNU, Luty, Zhou et al, BioRad, Wojno, De Boever, Sindhi, Maecker et al, ProtocolsOnline, and Dartmouth. The artisan would have been motivated to make and use the invention as claimed because Dartmouth teaches use of 1% formalin for delayed flow cytometric analysis. The artisan would have had a reasonable expectation of success based on the cumulative disclosures of these prior art references.
Claim(s) 18-19 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. (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); as cited on the 05/20/2021 IDS), BioRad (Sample Preparation Protocol, obtained from: https://web.archive.org/web/20170721082805/https://www.bio-rad-antibodies.com/flow-cytometry-sample-preparation.html), 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)), De Boever et al, Sindhi, Maecker et al, and ProtocolsOnline, as applied to claims 1, 3-9, 11, 13-14 and 16, above, in further view of eBioscience (Staining Cell Surface Antigens for Flow Cytometry, revised 9/11/2013), obtained from: https://medschool.cuanschutz.edu/docs/librariesprovider52/main-research/shared-resources/flow-cytometry/cell-analysis/cell-analysis-additional-resources/staining-cell-surface-antigens-for-flow-cytometry.pdf?sfvrsn=5a3b29b9_4#:~:text=prior%20to%20staining.-,3.,30%20minutes%20at%20room%20temperature.).
Regarding claims 18-19, as discussed above, the combination of CWNU, Luty, Zhou et al, BioRad, Wojno, De Boever, Sindhi, Maecker et al, and ProtocolsOnline teach claim 11, step 10, but do not teach incubation of the sample with primary antibody for 20 minutes. However, eBioscience teaches incubation for 20-30 minutes.
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 CWNU, Luty, Zhou et al, BioRad, Wojno, De Boever, Sindhi, Maecker et al, ProtocolsOnline, and eBioscience. The artisan would have been motivated to make and use the invention as claimed because One of ordinary skill in the art would have found it obvious, as of the filing date, to use a timeframe of 20-30 minutes for incubation (in the dark, cool environment of CWNU) as an art-taught timeframe for such incubation and would have found it obvious to use 20 minutes as the most time effective time of the 20-30 minute time frame. The artisan would have had a reasonable expectation of success based on the cumulative disclosures of these prior art references.
Claim(s) 1, 3-9, 11 and 13-16 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. (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)), BioRad (Sample Preparation Protocol, obtained from: https://web.archive.org/web/20170721082805/https://www.bio-rad-antibodies.com/flow-cytometry-sample-preparation.html), 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)), 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), ProtocolsOnline (Phosphate buffered saline, last updated 10/3/2016, obtained from: https://www.protocolsonline.com/recipes/phosphate-buffered-saline-pbs/), and 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), and CN 106771242 A, hereinafter referred to as ‘Shihezi University’(as cited on the 05//2021 IDS).
As noted above, claims 1, 3-9, 11, 13-14 and 16 are held to be taught by the combination of CWNU, Luty, W., Zhou et al, Biorad, De Boever et al, Wojno et al, Sindhi, ProtocolsOnline, and Maecker et al.
These references do not specifically teach centrifugation for 20 minutes. The addition of Shihezi University to the combined teachings enumerated above remedies this deficiency.
Regarding claims 13 and 15, note that Shihezi University teaches a method of preparing cells wherein, abdominal aorta peripheral blood is gathered, placed in 10g/LEDTA vacuum test tubes, peripheral blood is centrifuged, serum and haemocyte are separated, PBS solution is added in haemocyte, it is fully mixed, after dilution haemocyte is gently laid on equivalent lymphocyte separation medium, 1500r, 30min, then extract cloud in 1.5ml centrifuge tubes, 1800r, 6min, then abandon supernatant, and PBS solution is resuspended, then standby (see claim 1, for example).
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 CWNU,’ in view of Luty, W., Zhou et al, BioRad, Wojno et al, De Boever et al, Sindhi, ProtocolsOnline, Maecker et al, and Shihezi University. The artisan would have been motivated to make and use the invention as claimed because Wojno teaches centrifugation at 4 °C for 10 min at 1500 rpm (480 × g), then a time of 20 minutes falls within the art-taught range of 10-30 minutes. Accordingly, absent evidence of unexpected results tied to that specific number, choosing 20 minutes would have been an obvious matter of choice, which one of ordinary skill in the art may choose with a reasonable expectation of success in the aim of optimizing the methods of cell preparation for flow cytometry. 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 artisan would have had a reasonable expectation of success based on the cumulative disclosures of these prior art references.
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 1 (after the cover page) of 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.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
UCSF (Buffy Coat Collection Instructions, obtained from: https://web.archive.org/web/20180712164137/https://nrgbb.ucsf.edu/sites/rtog.ucsf.edu/files/wysiwyg/BuffyCoatOnlyInstructionsMarch2013.pdf; as presented in the Office Action dated 09/06/2024) teaches isolation of the buffy coat by collecting whole blood into a EDTA (anticoagulant) tube and centrifuging within one hour, removing the plasma layer, then aliquoting the buffy coat layer.
Yale School of Medicine (flow cytometry (FACS) staining protocol (Cell surface staining), obtained from: https://medicine.yale.edu/immuno/flowcore/protocols/analysis/; as presented in the Office Action dated 09/06/2024) teaches a method of preparing samples for flow cytometry comprising wash the cells (single cell suspension) and adjust cell number to a concentration of 1-5x106 cells/ml in ice cold FACS Buffer (PBS, 0.5-1% BSA or 5-10% FBS, 0.1% NaN3 sodium azide*). Do not add sodium azide to buffers if you are concerned with recovering cell function e.g. if cells are to be collected for functional assays. It inhibits metabolic activity. We recommend staining with ice cold reagents/solutions and at 4°C, since low temperature and presence of sodium azide prevent the modulation and internalization of surface antigens which can produce a loss of fluorescence intensity.
Shihezi University, noted above in the rejections of record, broadly teaches a method for analyzing T lymphocytes by means of a flow cytometer, comprising: centrifuging an in-vitro anticoagulant blood sample; separating serum and blood cells; adding a PBS solution to the blood cells and mixing same; adding same to an equal amount of lymphocyte separation solution, performing centrifugation, and taking a cloud mist thereof for centrifugation, so as to obtain buffy coat cells; adding a PBS solution for resuspension, and adding the suspension to a flow tube; adding a flow antibody labeling a flow cytometry subset; and incubating same in the dark at room temperature for 30 min for testing (see claim 1).
Zhou et al, noted above in the rejections of record, broadly teaches a method for analyzing T lymphocytes by means of flow cytometer, comprising: adding anticoagulant blood to lymphocyte separation solution and centrifuging same, taking buffy coat cells and washing same; re-suspending same by means of a PBS solution and counting same; adjusting the concentration thereof to be 5 x 106 /mL; adding the suspension into a flow tube; adding a flow antibody labeling a flow cytometry subset, mixing evenly, placing same at 25°C and incubating in the dark for 30 min; centrifuging same and discarding the supernatant; adding a PBS solution again for re-suspension; and after centrifuging twice, adding a PBS buffer solution for re-suspension so as to perform flow testing (see pages 4016-4018, especially section 1.4).
Marsh et al (Eur Respir J. 2018 Jan 25;51(1):1701214. doi: 10.1183/13993003.01214-2017) teach flow cytometric analysis of human blood samples using antibodies against CD3, CD4, CD8, and CD123.
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/Ashley Gao/
Examiner, Art Unit 1678
/GREGORY S EMCH/Supervisory Patent Examiner, Art Unit 1678