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 .
Election/Restrictions
Applicant has elected Group I, claims 1-18, drawn to a method of processing a tissue sample. Claims 19 and 20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected inventions, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on September 19, 2025.
Applicant's election with traverse in the reply filed on September 19, 2025 is acknowledged. The traversal is on the grounds that the searches are coextensive and would not be unduly burdensome to search all claims together.
This is not found persuasive because examiner was able to provide art which satisfied the limitations of the method claims without being able to satisfy the limitations of all the product claims thereby demonstrating that a search and/or examination burden exists between the restricted groups. Additionally, as described in the restriction requirement dated July 22, 2025, the groups have acquired a separate status in the art as evidenced by their separate classifications.
The requirement is still deemed proper and is therefore made FINAL.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Status of the Claims
Claims 1-20 are pending. Claims 19 and 20 are withdrawn. Claims 1-18 are examined on the merits.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. The instant application claims domestic benefit from U.S. provisional application 63/341327 filed on May 12, 2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on May 11, 2032 is in compliance with the provisions of 37 CFR 1.97 and has been considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Objections to the Specification
The disclosure is objected to because of the following informalities: p. 7 in reference to Figure 5, describes elements in the figures with colors (e.g., bicolor and blue). However, Applicant has not submitted drawings executed in color.
Furthermore, Applicant is reminded that color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Claim Objections
Claims 1-4 and 8 are objected to because of the following informalities: claims recite both “the tissue sample” and “the sample”, which appears to refer to the same tissue sample. For consistency, it is suggested that Applicant amend the claims to recite consistent use of “tissue sample” in order to remove inconsistent terminology usage. Appropriate correction is required.
Claim 8 is objected to because of the following informalities: claim 8 recites “the section” and “the sample”. For consistency, it is suggested that Applicant amend the claims to recite consistent use of “tissue section” in order to remove inconsistent terminology usage. Appropriate correction is required.
Claim 15 is objected to because of the following informalities: claim 15 appears to contain typographical errors. Claim 15 recites “expositing the tissue sample to vacuum conditions”. As the definition of “expositing” is “to explain in detail”, based on the instant specification, this appears to be a misspelling of “exposing”. Additionally, “a vacuum conditions” simultaneously recites a singular vacuum condition and multiple vacuum conditions. Appropriate correction is required.
Claim 16 is objected to because of the following informalities: claim 16 recites the “metho” of claim 8 which appears to be a misspelling of method. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-2 and 11-16 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the limitation "the planar surface" in line 6. There is insufficient antecedent basis for this limitation in the claim as there is no prior recitation of a planar surface. Appropriate correction is required.
Claim 2, which depends from claim 1, recites the limitation wherein step (a) is done by immersing the sample in a solution comprising…”. There is insufficient antecedent basis for this limitation as step (a) of claim 1 recites “permeating the tissue sample with monomers” and does not recite wherein the monomers are in a solution. Appropriate correction is required.
Claim 11, which depends from claim 8, recites the limitation “wherein in step (ii), the tissue section is denatured…”. Step (ii) of claim 8 recites “incubating the tissue section with acrylamide…” and does not recite a denaturing step. Step (iii) of claim 8 recites a treatment using a denaturing agent. Thus it is unclear whether claim 11 is intended to further limit step (ii) of claim 8 which recites incubating the tissue section with acrylamide or step (iii) of claim 8 which recites treating the sample with a denaturing agent. Appropriate correction is required.
Claim 12, which depends from claims 8 and 1, recites the limitation "the one or more antibodies". There is insufficient antecedent basis for this limitation in the claim as there is no prior recitation of one or more antibodies. Appropriate correction is required.
Claims 13 and 14, which depend from claim 12, are also rejected as incorporating a limitation from a rejected claim while failing to correct the deficiency. Appropriate correction is required.
Claims 15 and 16, which depend from claims 8 and 1, recite the limitation "the analysis" in lines 1. There is insufficient antecedent basis for this limitation in the claim as there is no prior recitation of analysis. Claim 8 recites “analyzing”. Appropriate correction is required.
Claim Rejections - 35 USC § 103
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-6 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Dewal et al. (US 2020/0354782 A1, hereafter “Dewal”) and Sandkuijl et al. (WO 2020/055813 A1, hereafter “Sandkuijl”).
With regard to claims 1-6 and 18, Dewal teaches a technique using expansion microscopy for imaging of formalin fixed paraffin embedded tissue samples wherein the tissue samples are prepared by permeating with a solution of monomers which are polymerized into swellable hydrogel which can expand upon addition of water (Para. [0022], lines 1-7). Dewal teaches wherein the monomer solution comprises sodium acrylate, acrylamide, and N,N-methylenebisacrylamide and polymerization is induced with ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) (Para. [0048], lines 7-12). Additionally, Dewal teaches wherein expanding the sample comprising the polyelectrolyte gel comprises dialyzing in water (Para. [0049]), which is considered to read on an aqueous medium. Further, Dewal teaches wherein the tissue sample can be transferred to a Bind-Silane treated glass bottom plate (Para. [0162], lines 23-24 see also [0164], [0177]), which is considered to reasonably read on a planar substrate comprising a positively charged surface which has been silanized. Further Dewal teaches wherein the “sample is digested, the gel is detached from the slide; the gel is the dialyzed to expand it, optionally followed by polyacrylamide embedding and imaging” (Para. [0177], lines 12-15), thus providing support for expansion of tissue prior to adhesion to a slide. Dewal teaches wherein the tissue section can be from a human (Para. [0052], lines 12-13).
Dewal does not teach wherein the expanded tissue sample on Bind-Silane treated glass bottom plate is dehydrated.
Sandkuijl teaches a systems and methods for performing imaging mass cytometry (Abstract) wherein tissue samples may first be prepared using expansion microscopy (Pg. 108, Pre-analysis sample expansion using hydrogels, 1st para., line 6) comprising a gelation step wherein tissue is infused with monomers including sodium acrylate, acrylamide, and N, N’-methylenebisacrylamide (Pg. 109, 3rd para., lines 1-4), treated with a homogenization agent (Pg. 109, 4th para., line 1), and expanded by dialyzing in a buffer or water (Pg. 110, 1st para., lines 1-2). Additionally Sandkuijl teaches that methods of preparation of tissue sample sections are well known in the field of immunohistochemistry and include dehydration steps (Pg. 77, 2nd para., lines 2-3) and wherein dehydration of sections during preparation of samples (Pg. 78, 1st para., lines 2-3) can be used in order to increase sensitivity of the analysis (Pg. 78, 1st para., lines 8-9).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to combine the dehydration step of tissue section preparation for imaging analysis as taught by Sandkuijl with the method of tissue preparation for expansion microscopy comprising permeating tissue with monomers, allowing monomers to polymerize, expanding the tissue by hydrating, and adhering the tissue sample to a positively charged surface as taught by Dewal. A skilled artisan would have been motivated to make this combination as they would have recognized that both Dewal and Sandkuijl teach tissue preparation for expansion microscopy and that Sandkuijl teaches wherein dehydrating the tissue sample can provide improvements in the subsequent analysis of tissue sections using imaging mass cytometry. One having ordinary skill in the art would have had a reasonable expectation of success as both Dewal and Sandkuijl teach techniques for expansion microscopy comprising similar steps including permeating tissue with monomers to generate a swellable gel and expanding the tissue by exposure to water or a buffer.
Claims 1-7 are rejected under 35 U.S.C. 103 as being unpatentable over Dewal et al. (US 2020/0354782 A1, hereafter “Dewal”) and Sandkuijl et al. (WO 2020/055813 A1, hereafter “Sandkuijl”) as applied to claims 1-6 and 18 above, and further in view of Ho and Bruttig (US 2009/0090022 A1).
With regard to claim 7, as detailed above, the combined teachings of Dewal and Sandkuijl teach a method of processing a tissue sample comprising contacting the tissue with monomers which polymerize to create a swellable hydrogel, expanding the tissue in an aqueous solution, adhering the tissue to a positively charged surface, and dehydrating the tissue in preparation for further analysis.
While Sandkuijl teaches that methods of preparation of tissue sample sections are well known in the field of immunohistochemistry and include dehydration steps (Pg. 77, 2nd para., lines 2-3) and wherein dehydration of sections during preparation of samples (Pg. 78, 1st para., lines 2-3) can be used in order to increase sensitivity of the analysis (Pg. 78, 1st para., lines 8-9), Sandkuijl does not teach wherein dehydration is done in a desiccation chamber.
Ho and Bruttig teach a desiccation chamber which can be used to dry biological materials while preserving material integrity and which may be used to prepare biological materials for further research applications (Abstract). Additionally Ho and Bruttig teach that the desiccation chamber can be used to dehydrate biological materials (Para. [0009], line 6) including various tissues (Para. [0052], lines 3-4).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, that the teachings of Sandkuijl wherein tissue samples are dehydrated prior to subsequent analysis could be substituted with the teachings of Ho and Bruttig wherein a desiccation chamber can be used to dehydrate tissue prior to use in subsequent analysis with the predictable result of generating a dehydrated tissue sample. One having ordinary skill in the art would have had a reasonable expectation of success as both Sandkuijl and Ho and Bruttig teach dehydration of tissue samples.
Claims 1-6 and 8-18 are rejected under 35 U.S.C. 103 as being unpatentable over Dewal et al. (US 2020/0354782 A1, hereafter “Dewal”) and Sandkuijl et al. (WO 2020/055813 A1, hereafter “Sandkuijl”) as applied to claims 1-6 and 18 above, and further in view of Ku et al. (2016. Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nature biotechnology, 34(9), 973-981; found in IDS; hereafter “Ku”) and Vaughan et al. (US 2017/0276578 A1, hereafter “Vaughan”).
With regard to claims 8 and 11, as detailed above, the combined teachings of Dewal and Sandkuijl teach a method a processing tissue comprising contacting the tissue with monomers which polymerize to create a swellable hydrogel, expanding the tissue in an aqueous solution, adhering the tissue to a positively charged surface, and dehydrating the tissue in preparation for further analysis. Dewal teaches wherein the tissue sample is a formalin fixed paraffin embedded tissue sample adhered on a slide (Para. [0010], lines 2-4) and wherein tissue can be treated with heat (Para [0116]) in order to increase antigen immunoreactivity lost during fixation of the tissue due to methylene bridge formation (Para. [0115], lines 1-5). In regard to the order of events, Dewal teaches this antigen retrieval heating step is after deparaffinization and before monomer polymerization [0159-0160]. Dewal teaches wherein the tissue is stained for targets of interest (Para. [0022], lines 10-11) which can be detected using one or more antibodies (Para. [0010], line 39), which is considered to read on one or more binding agents, linked to a detectable label (Para. [0010], lines 42-43) wherein the label can be a pigment, dye, or other chromogen (Para. [0137], lines 6-7). Dewal further teaches use of imaging of targets via confocal microscopy or super-resolution microscopy of RNA using multiplexed fluorescence in situ hybridization (FISH) and hybridization chain reaction (Para. [0133]), which is considered to read on analyzing the binding of the binding agents to the tissue section.
The combined teachings of Dewal and Sandkuijl do not teach incubation of the tissue section with acrylamide to quench methylols nor treating the sample with a “denaturing agent”. However, Dewal teaches treatment of tissue samples with a protease (Para. [0042, line 14) and Sandkuijl teaches treatment of a homogenizing agent which allows for sample expansion and which can be a protease (Pg. 109, 4th para. Line 4) and further cites Vaughan (US 2017/276578) (Pg. 110, line 2).
Ku teaches method for super resolution imaging of tissues wherein the tissue is expanded while preserving overall architecture and cellular organizations of proteins (i.e., expansion microscopy) wherein crosslinking of proteins during hydrogel tissue expansion is prevented, allowing for tissue expansion during denaturation (Abstract). Ku teaches improvements to expansion microscopy techniques (Pg. 973, right columns) comprising a step of incubating tissue in a high concentration of 20% acrylamide for 1 hour during the hydrogel-tissue hybridization in order to quench reactive methylols formed by interaction of proteins and tissue fixative, thus preventing the protein crosslinking (Pg. 974, right column, 1st para.) which limits subsequent tissue expansion (Pg. 975, left column, lines 4-7) and allowing for greater tissue expansion (Figure 1). Ku additionally teaches subsequent antibody staining of the expanded tissue for visualization of proteins (Figure 2).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention to combine the teachings of Dewal and Sandkuijl wherein a formalin fixed tissue sample is contacted with monomers which are allowed to polymerize to form a hydrogel which can be subsequently expanded in an aqueous medium with an acrylamide incubation step to quench methylols as taught by Ku. A skilled artisan would have been motivated to combine the acrylamide incubation step of Ku with the combined teachings of tissue preparation as taught by Dewal and Sandkuijl because Ku teaches that incubation with a high concentration of acrylamide quenches reactive methylols and allows for increased tissue expansion and thus better visualization and spatial organization of the proteins in the tissue. One having ordinary skill in the art would have had a reasonable expectation of success as Dewal, Sandkuijl, and Ku all teach methods of tissue expansion prior to antibody staining.
Vaughan teaches a method of preparing a sample for expansion microscopy comprising permeating a fixed tissue sample with monomers, polymerizing the monomers to create a water swellable composition, treating the tissue sample with a homogenizing agent, and then dialyzing the sample in water to cause expansion (Para. [0008], lines 7-20). Additionally, Vaughan teaches wherein the homogenizing agent is used to allow for sample expansion (Para. [0068], line 6) and wherein the sample can be homogenized by denaturation with SDS (Para. [0068], lines 13-14), which is considered to read on treatment of the tissue sample with a denaturing agent.
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to combine the teachings of Sandkuijl the tissue sample is treated with a homogenizing agent to allow for sample expansion with the teachings of Vaughan wherein a tissue sample is treated with a homogenizing agent to allow for sample expansion and wherein denaturation with SDS can be used as the homogenizing agent. A skilled artisan would have recognized that it is predictably obvious to substitute the homogenizing agent which can be a protease as taught by Sandkuijl with the homogenizing agent which can be a denaturing agent (i.e., SDS) as taught by Vaughan. One having ordinary skill in the art would have had a reasonable expectation of success as Dewal, Sandkuijl, and Vaughan all teach tissue preparation techniques for expansion microscopy.
In regard to the order of the steps, although the combined teachings of Dewal, Sandkuijl, Ku, and Vaughan do not explicitly state the order, they disclose the steps individually, and their combination into the claimed order would have been obvious and would have been recognized to achieve a similar results. See Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.).
With regard to claim 9, Dewal teaches wherein tissue samples can be heated for 5-40 minutes at 90-100°C (Para. [0116], lines 7-9).
With regard to claim 10, as stated above, Ku teaches wherein tissue is incubated with 20% acrylamide for 1 hour (Figure 1 and Pg. 976, left column, 1st para. lines 2-3).
With regard to claims 12-16, as detailed above, Dewal teaches staining of the tissue section (Para. [0022], lines 10-11) via binding of one or more antibodies linked to a detectable label (Para. [0010], lines 39 and 42-43) prior to use of analysis of the binding using via confocal microscopy or super-resolution microscopy of RNA using multiplexed fluorescence in situ hybridization (FISH) and hybridization chain reaction (Para. [0133]).
Dewal does not teach wherein the one or more antibodies are an antibody cocktail, which contains at least 10 antibodies which are mass tagged nor does Dewal teach wherein the analysis comprises exposure of the sample to vacuum conditions and wherein analysis is done by multiplexed ion beam imaging, imaging mass cytometry, or co-detection by indexing.
Sandkuijl teaches use of expansion microscopy for use in preparation of tissue samples (Pg. 108, Pre-analysis expansion using hydrogels and 1st para., line 6) and methods for analysis of samples using imaging mass cytometry (Abstract). Sandkuijl teaches a method wherein, like in FISH, “specific binding partners (SBPs)” such as antibodies comprising detectable labels are attached to targets in the sample (Pg. 3, 1st para., lines 8-10) and wherein the imaging mass cytometry technique allows for multiplexed analysis of many labels in parallel (Pg. 3, 1st para., lines 16-18). Sandkuijl teaches wherein the sample can be contacted with one or more tagged antibodies and wherein 10 or more antibodies may be used (Pg. 98, 2nd para., lines 2-5), thus providing support for use of an antibody cocktail comprising at least 10 antibodies. Further, Sandkuijl teaches wherein a labeled SBP is an antibody wherein the labeling can be achieved through attachment of a mass tag (Pg. 88, Antibody SBP members, 1st para., lines 1-3). Additionally, Sandkuijl teaches use of electron microscopy for analysis of tissue samples (Pg. 74, 2nd para., line 1) which have been stained with heavy atom labels analysis (Pg. 74, 3rd para., lines 6-7) wherein the tissue sample is held under vacuum conditions (Pg. 74, 2nd para., line 5).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to combine the teachings of Dewal wherein one or more antibodies are used as binding agents of the tissue section prior to analysis using microscopy techniques with the teachings of Sandkuijl wherein more than 10 mass tagged antibodies are used as binding agents of the tissue section prior to analysis using imaging mass cytometry which can be complexed with electron microscopy. Dewal teaches embodiments wherein the antibodies comprise fluorescent dyes but that detection of target molecules is limited based on the small number of fluorescent dyes and by dampening of the fluorescent signal due to polymerization of the monomers (Para. [0005], lines 18-23). Sandkuijl teaches wherein mass tagged antibodies in conjunction with imaging mass cytometry provides an advantage of fluorescent microscopy because the signals do not overlap which enables simultaneous imaging of 40 or more proteins in a tissue section (Pg. 78, 3rd para., lines 1-3). Therefore, a skilled artisan would have been motivated to combine the teachings of Dewal and Sandkuijl in order to allow for more specific and robust imaging and analysis techniques via use of mass tagged antibodies and imaging mass cytometry. One having ordinary skill in the art would have had a reasonable expectation of success as both Dewal and Sandkuijl teach use of antibody binding and imaging techniques in tissue samples prepared using expansion microscopy techniques.
With regard to claim 17, Dewal teaches wherein the tissue section is paraffin embedded which is deparaffinized (Para. [0010], lines 2-5).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERIN V PAULUS whose telephone number is (571)272-6301. The examiner can normally be reached Mon-Fri 8 AM-5 PM.
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/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631