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 .
DETAILED ACTION
Response to Amendment and Arguments
The Amendment filed on 10/20/2025 in response to the previous Non-Final Office Action (6/18/2025) is acknowledged and has been entered.
Claims 4, 11-15, 30, and 36-40 have been cancelled.
Claims 1-3, 5-10, 16-29 and 31-35 are pending and examined for a method of analyzing liquid sample and for the presence of rare circulating cells on merits.
The following office action contains NEW GROUNDS of rejection-based on the amendment.
Rejections/Objection Withdrawn
The improper Markush Grouping rejection of Claims 8, 10, and 26-27 is withdrawn in view of claim amendment.
The rejections of Claims 24 and 32 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph are withdrawn in view of the amendment to the claims.
The rejection of Claim 21 under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph is withdrawn in view of the amendment to the claim.
The rejections of under 35 U.S.C. 102(a)(2) as being anticipated by T. Tafas (WO2022/155213) as T.Tafas-WO or T. Tafas (US2021/0018441) as Tafas-USPub are withdrawn in view of amendment to the claims and applicant arguments.
The rejections of Claim(s) under 35 U.S.C. 103 as being unpatentable over T. Tafas (WO2022/155213) and/or Tafas (US2021/0018441) in view of Ulmer and Boothe et al are withdrawn in view of amendment to the claims.
The arguments are moot in view of withdrawals of the rejection(s).
New GROUND Rejection-base on the claim amendment
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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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.
1. Claims 1-3, 5, 8-10, 16-21, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over T. Tafas (US2021/0018441, published Jan 2011 and filed on June 2017) in view of Boothe et al (eLIFESciences, Tools and Resources, 2017, e27240). All the references provided in the last Office action.
Tafas discloses a system/method of detecting Circulating Tumor Cells (CTC) from liquid sample comprising biological materials for target components, wherein the liquid sample is liquid biopsy and while blood cells from blood samples. The method comprises: preparing sample comprising biological materials from liquid biopsy or blood sample comprising lysing red blood cells [0157] and obtained sample comprising CTC the T-cells [0010, 0088], immunostaining the presence of antigens on the cells including CTC) in the samples with fluorescently labelled antibodies, wherein the antibodies against an epithelial cell antigen EpCAM etc. selectively on CTC and CD45 for leukocytes common antigen are included [0157, 0176], and then mixing the counterstained cells with low melting point agarose, or collagen, or polyacrylamide to allow to solidify and form hydrogel sample fixture (gelation), which is mounted on specimen holder [0103, 0139, 0163], and analyzing/Imaging the solidified sample with fluorescent microscopy scanning [0087, 0170]. Tafas teaches using selective plane illumination microscopy (SPIM), also known as light sheet fluorescence microscopy (LSFM), which could scan fluorescence labeled sample with high resolution and minimal photo bleaching.
Tafas does not teach a refractive index matching material introduced into the solidified sample hydrogel sample recited in the amended claims 1.
Boothe et al teach introducing Lodixanol, a new and specific refractive index matching medium (RIM), to a live imaging cells or tissue specimen to improve image quality in primary cell culture or organoids, organism and cells immobilized in agarose for live cell imaging (abstract and entire document, figure 2 in particular). Boothe et al also teach that modulation for the specimen’s osmolality and optical clarity in hydrogen formation by adjusting pH with buffering (page 4). Boothe et al, at page 2, last para, specifically teach:
As Iodixanol is highly water-soluble, simple dilution into aqueous solutions can be used to linearly tune the refractive index of the solutions between RI 1.333 – RI 1.429 (Figure 1a). For water, PBS and culture media of aquatic model organisms, the medium only minimally affected the refractive index at a given Iodixanol concentration….
……Organisms are often immobilized in agarose for live imaging. Agarose polymerization was not prevented at any Iodixanol concentration and agarose concentrations in ranges used for specimen immobilization did not significantly affect the refractive index of Iodixanol solutions (Figure 1c), thus making Iodixanol compatible with agarose embedding protocols. A further important requirement especially for fluorescence-based live imaging applications is low autofluorescence…..
Legend of Figure 1. Physicochemical properties of the refractive index matching agent Iodixanol. (a). Solvent dependency of the refractive index of Iodixanol. (b) Temperature dependency of the refractive index of Iodixanol solutions. Water was used as a solvent. (c) The refractive index of Iodixanol gels at various agarose concentrations. (a–c) Inset diagrams show a magnified region of the respective data set. Measurements were taken at 10% Iodixanol concentration increments as technical triplicates.
Thus, Boothe et al teach and suggest that lodixanol as a new and specific refractive index matching material can be introduced into hydrogel (agarose) solidified sample with live cells to obtain an optically cleared solidified sample.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention was made to prepare sample comprising biological materials from liquid biopsy, which is mixed with solidifying agent (hydrogel) and RIM for getting better imaging of rare circulating cells in the solidified specimen with expected result. In order to obtain and improve the image of the cells in the hydrogel specimen, one of ordinary skill in the art before the effective filing date of was made would have been motivated with reasonable expectation of success to introduce the RIM medium to hydrogel specimen and image the cells which are stained with fluorescent labeled antibody because Tafas has shown the method of obtaining the solidified specimen from liquid biopsy, which has been stained with fluorescent labeled antibodies, Boothe et al have shown an improved method of microscopy imaging by adding RIM medium to live cell, organism and cells immobilized in agarose (one form of hydrogel). Therefore, the references in combination teach and suggest each and every limitation of the invention before the effective filing date of the invention was made, absent unexpected result.
2. Claims 1-3, 5, 6-7, 8-10, 16-21, 23, 24-29, and 31-35 are rejected under 35 U.S.C. 103 as being unpatentable over T. Tafas (US2021/0018441, published Jan 2011 and filed on June 2017) and Boothe et al (eLIFESciences, Tools and Resources, 2017, e27240) as set forth above, further in view Ulmer et al (Immunobiology, 166: 238-250, 1984, abstract only). All the references provided in the last Office action.
The teachings of Tafas and Boothe et al are set forth above, the references do not the specimen from liquid blood sample and being peripheral blood mononuclear cells (PBMC, claims 6-7 and 24-29, and 31-35).
Isolating PBMC from blood is well known technology and has been used for many decades and the technology has been improved with time. Ulmer et al teach a method isolating PBMC from blood by density gradient centrifugation on Percoll that is an improved method and achieves better separation for mononuclear cells (lymphocytes and monocytes) as compared to the method of isolating PBMC by Ficoll-isopaque (abstract).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention was made to prepare sample comprising biological materials from liquid biopsy or blood PBMC and a refractive index matching material (RIM) for getting better imaging of live cells in the solidified specimen with expected result. In order to image the circulating cell isolated from blood one skilled in the art would be motivated with reasonable expectation of success to isolate PBMC and rare cells from blood and mix them with hydrogel to form a solidified specimen with refractive index matching material because Tafas has shown the method of obtaining the specimen from liquid biopsy and stained with fluorescent labeled antibodies, Boothe et al have shown an improved method of microscopy imaging by adding RIM medium to live cell, organism and cells immobilized in agarose (one form of hydrogel) and Ulmer et al have shown an improved method isolating cells and PBMC from blood. Therefore, the references in combination teach and suggest each and every limitation of the invention before the effective filing date of the invention was made, absent unexpected result.
3. Claims 1-3, 5, 8-10, 16-21, 22, and 23 are rejected 35 U.S.C. 103 as being unpatentable over are rejected under 35 U.S.C. 103 over T. Tafas (US2021/0018441, published Jan 2011 and filed on June 2017), Boothe et al (eLIFESciences, Tools and Resources, 2017, e27240) as set forth above, further in view of Kirsch notes (EMS Microscopy Academy, published 2018). All the references provided in the last Office action.
The teachings of Tafas, and Boothe et al on the method of detecting CTC cell or blood cells are set forth above. none of the references teaches fixation of the specimen with glutaraldehyde or formaldehyde set forth in claim 22.
Kirsch-Notes teaches a method of chemical fixation for biological sample for light and electron microscopy, which includes using paraformaldehyde and glutaraldehyde to fix live cells as well as concentration of the reagents etc. and teach that the fixation is required to halt biochemical reaction, prevent autolysis, stabilize macromolecules and preserve delicate morphologies (entire document).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention was made to fix the specimen for the imaging of circulating cells with microscopy with expected result. In order to improve the process of imaging the rare circulating cells in the hydrogel specimen, one of ordinary skill in the art before the effective filing date of was made would have been motivated with reasonable expectation of success to fix the specimen with fixative solution including glutaraldehyde and/or formaldehyde before the imaging with microscopy because Tafas and Boothe et al have shown the method of making the specimen to obtain an optically cleared solidified sample for imaging and Kirsch-Notes has shown requirement of fixation for biological samples for light microscopy to prevent autolysis, stabilize macromolecules and preserve delicate morphologies etc. (entire document). Therefore, the references in combination teach and suggest each and every limitation of the invention before the effective filing date of the invention was made, absent unexpected result.
Response to applicant’s argument:
Based on the claim amendment, the rejections have been newly formed with different combination of the references and involved in different claims, applicant's argument will be responded accordingly as follows:
Tafas I (Pg-publication):
At page 1, applicant agrees that Tafas I teaches all the limitation for the independent claims 1 and 24 except no RM matching material (claims 1 and 24) and no PBMC (claim 24).
In response, the deficiency has been made up with the reference by Boothe, and further by Ulmer as addressed above in the newly formed rejections.
Boothe et al:
At page 14, applicant argues:
Boothe discloses "lodixanol as a non-toxic medium supplement that allows refractive
index matching in live specimens" and describes lodixanol as "commercially available under the brand name OptiPrepTM" with "a high refractive index of 1.429 as a 60% stock solution", but this disclosure is fundamentally directed to live imaging applications in mounting media, not to introducing refractive index matching material to already solidified samples.
Applicant cites eLide digest (last paragraph) as
It is important to stress that lodixanol does not change the
refractive index of the sample or cancel out refractive index
differences within the sample-so it cannot render opaque
backspace opaque specimens transparent. Nevertheless,
lodixanol supplementation is a simple and affordable technique to
improve image quality in any live imaging application without
having to resort to more expensive and highly specialized
microscopes
Boothe acknowledges the limitations of its approach, stating that "lodixanol does
not change the refractive index of the sample or cancel out refractive index differences within the sample - so it cannot render opaque specimens transparent", which is fundamentally incompatible with the claimed method that requires "introducing a refractive index matching material to the solidified sample to obtain an optically cleared solidified sample" as recited by amended claims 1 and 24.
To the contrary, under Boothe's method "simple dilution into aqueous solutions can be
used to linearly tune the refractive index of the solutions ….in mounting media before solidification - which is the opposite of the instantly claimed approach of introducing refractive index matching material to an already solidified sample.
Boothe acknowledges that "lodixanol supplementation cannot correct for are refractive index differences within the specimen" and "cannot achieve the in-toto RI matching of fixed tissue protocols" (Boothe, page 8 of 15), demonstrating that Boothe's live imaging approach is fundamentally different from the claimed matrix-assisted method that provides
optically cleared solidified samples. Instead, Boothe's work is specifically directed toward "developing an RI matching medium for live-imaging" with requirements for "high water solubility as prerequisite for dilution into regular culture media" and "low toxicity as crucial requirement for live-imaging compatibility" (Boothe, pages 1-2), which are concerns specific to live cell imaging that do not apply to the claimed method of analyzing solidified samples.
In response, the Office disagrees:
1) the instant claims broadly encompass any refractive index matching material to solidified sample to obtain an optically cleared solidified sample. Boothe’s lodixanol meets the requirement as taught:
Organisms are often immobilized in agarose for live imaging. Agarose polymerization was not prevented at any Iodixanol concentration and agarose concentrations in ranges used for specimen immobilization did not significantly affect the refractive index of Iodixanol solutions (Figure 1c), thus making Iodixanol compatible with agarose embedding protocols (page 2, last paragraph).
Thus, Boothe teaches live cells or tissues (organisms) mixed into solidified specimen with iodixanol, a new RIM, immobilized in agarose.
2) as discussed in the rejection, Iodixanol is highly water-soluble, simple dilution into aqueous solutions. Agarose polymerization was not prevented at any Iodixanol concentration, which indicates that iodixanol can be applied at any stage of the specimen preparation, before or after cell-agarose gel solidified into specimen.
3) For function of Iodixanol as refractive index matching material for optically cleared solidified sample as instantly claimed, Boothe teaches imaging of specimen with Iodixanol significantly improve the clarity of the cells in the images, see figure 3-4, for results.
For the reasons, the references of Tafas I in combination of Boothe teaches claimed method set forth in claim 1 and part of claim 24 (not PBMC, see rejections).
Ulmer and Kirsch et al:
At page 13, paragraph 5, and page 16, applicant argues:
Ulmer does not provide disclosure related to refractive index matching or optical clearing of solidified samples (page 13).
Kirsch does not provide disclosure related to refractive index matching or optical clearing of solidified samples (page 16).
In response, the Office made the rejection under 35 U.S.C. 103(a) is based on the guideline of MPEP 2141 (rejection under 35 USC 103), particularly, MPEP 2141.02, which states
In determining the difference between the prior art and the claims, the question under 35 USC103 is not whether the difference themselves would have been obvious, but whether the claimed invention as a whole would have been obvious.
It is improper to argue and discuss the references cited under USC 103 rejected individually without clearly addressing the combined teachings. It must be remembered that the references are relied upon in combination and are not meant to be considered separately. Thus, in this case, as stated in the rejection above,
Tafas I teaches general method of analyzing a sample comprising live cells, Boothe teaches refractive index matching material for the optically cleared solidified sample, and Ulmer teaches the live PBMC obtained from blood, Kirsch teaches the sample fixation procedure. The references in different combinations would teach and suggest each and every limitation set forth in different claims.
For all the reasons above, the rejections are made again with the same references and new format.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Lei Yao, whose telephone number is (571) 272-3112. The examiner can normally be reached on 8:00am-6:00pm Monday-Friday.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Samira Jean-Louis, can be reached on (571) 270-3503. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/LEI YAO/ Primary Examiner, Art Unit 1642
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