NOTE: The examiner for this Office Action has changed. The new Examiner is Sean Aeder.
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 5/15/26 has been entered.
Claims 1-3, 5-10, 16-29, and 31-35 are pending.
Claims 1 and 24 have been amended by Applicant.
Claims 1-3, 5-10, 16-29, and 31-35 are currently under consideration.
This Office Action contains New Rejections Necessitated by Amendments.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Interview Request
An Automated Interview Request (AIR) was submitted by Applicant on 5/28/26, requesting an interview between 6/25/26-6/30/26.
Applicant was subsequently notified the examiner for the application had changed.
After the application was docketed to the current examiner, the current examiner emailed Jason Chumney 7/1/26 proposing interview dates between 7/2/26-7/7/26.
On 7/24/26, Jason Chumney replied to the current examiner’s email with an email and phone call and proposed an interview “early September”.
On 8/3/26, the current examiner replied to Jason’s 7/24/26 email and phone call and left a message to set up an interview time.
On 8/18/26, Jason telephoned the examiner and reiterated interest in conducting an interview. However, Jason was unable to commit to an interview time. During the telephone call, the current examiner notified Jason that the examiner has limited time to reply to the RCE that was submitted prior to the AIR.
On 9/25/26, the current examiner left a voicemail message with Jason that any interview prior to another Office Action will need to be conducted before 9/29/26 and requested a call-back to set-up an interview.
The examiner waited until 5pm ET on 9/29/26 to approve this Office Action for mailing.
If applicant desires an interview in response to this Office Action, applicant is invited to contact the examiner and proposed a date and time for an interview.
Rejections Withdrawn
All previous rejections are withdrawn.
New Rejections Necessitated by Amendments
Claim Rejections - 35 USC § 112
Claims 24-29 and 31-35 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.
Claims 24-29 and 31-35 are rejected because step “(c)” of claim 34 recites “…wherein the biological materials have….”. There is insufficient antecedent basis for “the biological materials” in the claims.
Claim Rejections - 35 USC § 103
Claims 1-3, 5-10, 16-19, 23-29, and 31-35 are rejected under 35 U.S.C. 103(a) as being unpatentable over Tafas et al (US 2021/0018441 A1; 1/21/21) in view of Boothe et al (eLIFE, 2017, e272240, 1-15), Ghigo et al (Methods in Molecular Biology vol 1763, Humana Press, NYC, pages 53-61), Chakarov et al (Science, 2019, 363 (1190): 1-18), Chi et al (J Vis Exp, 2018, 137(e58271): 1-9), and Matsushiro et al (Biotechnology and Bioengineering, 2018, 115: 1614-1623).
Tafas et al teaches a method of detecting target cells in a liquid blood sample that includes: (i) obtaining a blood sample from a subject, (ii) centrifugation to separate cell layers from blood serum and removal of a cell layer (as defined by the instant specification at lines 18-20 of page 19, such a centrifugation + removal step is of Tafas et al is a step of “enriching” a biological liquid sample by concentrating from a larger volume of the liquid sample), (iii) suspending the removed cell layer of (ii), (iv) immobilization cells of the removed layer, and (v) immunostaining of the immobilized layer and subjecting the immobilized cells to selective plane illumination microscopy (SPIM; “LSFM”) to visualize the immunostained solidified sample ([0019]-[0031], in particular).
For convenience, all steps outlined in [0019]-[0031] of Tafas et al are set forth below:
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Tafas et al further teaches the immobilization is in a gel that maintains cell structure, such as agaraose gel, or a collagen, or a polyacrylamide gel ([0037], in particular). For detecting tumor cells as the target cells in a blood sample comprising white blood cells (which include T-cells; see [0044]) and tumor cells, Tafas et al teaches preparing a sample comprising lysing red blood cells in the sample ([0157], in particular), resulting in obtaining a sample comprising CTCs and T-cells ([0010] and [0088], in particular). At [0157] and [0176]. Tafas et al further teaches using fluorescently-labeled antibodies to differentially immunostain the resulting sample for cells expressing CTC or leukocyte antigens (including EpCAM on CTCs and CD45 on leukocytes - including T cells, B cells, NK cells, monocytes, and granulocytes) and mixing the stained cells with a low melting point agarose, or collagen, or polyacrylamide matrix and allowing the mixture to solidify/immobilize and form a hydrogel sample fixture (gelatin) – which is mounted on a specimen holder and analyzed/imaged with fluorescent scanning microscopy ([0103], [0139], [0163], [0087], and [0170], in particular). At [0170], Tafas et al further teaches using selective plane illumination microscopy (SPIM; “LSFM”) to visualize the immunostained solidified sample.
Tafas et al does not specifically teach contacting a solidified hydrogel fixture with a refractive index matching material to permit the refractive index matching material to diffuse into a solidified sample to produce an optically clearer solidified sample. However, these deficiencies are made up in the teachings of Boothe et al, Ghigo et al, Chakarov et al, Chi et al, and Matsushiro et al.
Boothe et al teaches refractive index mismatches between tissue and surrounding medium results in aberrations that distort microscopic imaging, which can be overcome using optical clearing techniques that equilibrate refractive index heterogeneity within samples (Introduction on page 1, in particular). Boothe et al further teaches clarifying biological samples that are subsequently immobilized in agarose with the refractive index matching clarifying solution Iodixanol, under conditions sufficient to permit Iodixanol to diffuse into the immobilized/solidified samples, to optically clarify prior to imaging the stained samples (Abstract, in particular). Boothe et al further teaches Iodixanol as “compatible with agarose embedding protocols” at page 2.
Ghigo et al and Chakarov et al both teach agarose embedding protocols comprising incubating biological samples solidified in agarose in the refractive index matching clarifying solution Rapiclear 1.47, under conditions sufficient to permit Rapiclear 1.47 to diffuse into the solidified samples, to optically clarify after immunologically staining the samples and prior to imaging the stained samples (steps 15 and 16 on page 58 of Ghigo et al and page 10 of Chakarov et al, in particular). Ghigo et al further teaches the samples are mounted on specimen holder that is a “vibratome” (step 1 on page 54, in particular) – which is capable of shaking/vibrating.
Chi et al teaches an agarose embedding protocol comprising incubating biological samples that comprise blood vessels solidified in agarose, while shaking, in the refractive index matching clarifying solution dibenzyl ether (DBE), under conditions sufficient to permit DBE diffuse into the /solidified samples, to optically clarify after immunologically staining the samples and prior to imaging the stained samples (page 3, in particular).
Matsushiro et al teaches an agarose embedding protocol comprising incubating fixed biological samples immobilized/solidified in agarose in the refractive index matching clarifying solution SCALEVIEW-A2, under conditions sufficient to permit SCALEVIEW-A2 to diffuse into the fixed solidified samples, to optically clarify after immunologically staining the samples and prior to imaging the stained samples (left column on page 1617, in particular).
One of ordinary skill in the art would have been motivated, with a reasonable expectation of success, to perform a combined method of analyzing a blood sample to detect circulating tumor cells (CTCs) comprising performing a method of Tafas et al comprising detecting target cells that are CTCs in a liquid blood sample comprising CTCs and PBMCs by: (i) obtaining a blood sample from a subject and lysing the red blood cells in the sample (as taught by Tafas et al), (ii) centrifuging the sample to separate cell layers from blood serum (which would also remove platelets) and removing a cell layer, (iii) suspending the removed cell layer of (ii), (iv) solidifying cells of the removed layer in low melting point agarose, collagen, and/or polyacrylamide matrix of Tafas et al and allowing the mixture to solidify/immobilize and form a hydrogel sample fixture (gelatin) that is mounted on a specimen holder of Tafas et al, (v) immunostaining the immobilized and solidified cells with in the specimen holder with fluorescently labeled antibodies of Tafas et al specific for leukocytes (PBMCs) and fluorescently labeled antibodies of Tafas et al specific for CTCs, (vi) immersing the immunostained samples immobilized in agarose in the specimen holder in a refractive index matching clarifying solution (of Ghigo et al, Chakarov et al, Chi et al, and/or Matsushiro et al) under conditions sufficient to permit the clarifying solution to diffuse into the immobilized and solidified samples to optically clarify the stained samples, and (vii) subjecting the immobilized cells to fluorescent scanning microscopy and/or selective plane illumination microscopy (SPIM; “LSFM”) to visualize the immunostained solidified sample for the presence of CTCs because (i) Boothe et al teaches refractive index mismatches between tissue (comprising cells) and surrounding medium results in aberrations that distort microscopic imaging, which can be overcome using optical clearing techniques that equilibrate refractive index heterogeneity within samples (Introduction on page 1, in particular) and (ii) Ghigo et al, Chakarov et al, Chi et al, and Matsushiro et al all each teach agarose embedding protocols comprising incubating fixed biological samples solidified in agarose in a refractive index matching clarifying solution, under conditions sufficient to permit refractive index matching clarifying solution to diffuse into the fixed immobilized/solidified samples, to optically clarify after immunologically staining the samples and prior to imaging the stained samples. This is an example of (i) some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention and/or (ii) applying a known technique to a known method ready for improvement to yield predictable results. See MPEP 2143.
In particular regards to instant claim 3, one of ordinary skill in the art would have been motivated, with a reasonable expectation of success, to perform the combined method wherein the immunostaining is performed after (as taught by Ghigo et al, Chakarov et al, Chi et al, and Matsushiro et al) or before (as taught by Boothe et al) immobilizing the sample in order to stain live cells because the prior art teaches successfully immunostaining before or after immobilization. In particular regards to instant claim 31, one of ordinary skill in the art would have been motivated, with a reasonable expectation of success, to perform the combined method wherein the low-melting point agarose is first added to an immunostained sample above room temperature (37 °C, as taught by [0163] Tafas et al) and then allowed to achieve reduced temperature at or below room temperature because those skill in the art know that is how low-melting point agarose solidifies.
In particular regards to instant claim 16, one of ordinary skill in the art would have been motivated, with a reasonable expectation of success, to perform the combined method wherein the specimen holder in which the samples is immobilized in agarose is a vibratome of Ghigo et al that shakes the sample when immerged in the clarifying solution because Ghigo et al teaches samples are mounted on specimen holder that is a “vibratome” (step 1 on page 54, in particular), Chi et al teaches samples immobilized and solidified in agarose are shaken when immersed in clarifying solution (page 3, in particular), a vibratome is clearly capable of shaking, and shaking would help mix and disperse the clarifying solution in which the immobilized and solidified in agarose are incubated.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art, absent unexpected results.
Claim Rejections - 35 USC § 103
Claim(s) 1-3, 5-10, 16-29, and 31-35 is/are rejected under 35 U.S.C. 103 as being unpatentable over Tafas et al (US 2021/0018441 A1; 1/21/21) in view of Boothe et al (eLIFE, 2017, e272240, 1-15), Ghigo et al (Methods in Molecular Biology vol 1763, Humana Press, NYC, pages 53-61), Chakarov et al (Science, 2019, 363 (1190): 1-18), Chi et al (J Vis Exp, 2018, 137(e58271): 1-9), and Matsushiro et al (Biotechnology and Bioengineering, 2018, 115: 1614-1623), as applied to claims 1-3, 5-10, 16-19, 23-29, and 31-35 above, and further in view of Kirsch-Notes (EMS Microscopy Academy, published 2018).
Tafas et al, Boothe et al, Ghigo et al, Chakarov et al, Chi et al, and Matsushiro et al are discussed above.
Tafas et al, Boothe et al, Ghigo et al, Chakarov et al, Chi et al, and Matsushiro et al do not specifically teach the combined method wherein the biological sample is fixed by incubating the sample in a fixative solution comprising glutaraldehyde, formaldehyde, or an epoxy. However, these deficiencies are made up in the teachings of Kirsch-Notes.
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, and teaches that the fixation is required to prevent autolysis, stabilize macromolecules and preserve delicate morphologies (entire document).
One of ordinary skill in the art would have been motivated, with a reasonable expectation of success, to perform the combined method wherein the biological sample is fixed using paraformaldehyde and glutaraldehyde of Krsch-Notes in an effort to prevent autolysis, stabilize macromolecules and preserve delicate morphologies because 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, and teaches that the fixation is required to prevent autolysis, stabilize macromolecules and preserve delicate morphologies (entire document).
This is an example of some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. See MPEP 2143. Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art, absent unexpected results.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SEAN E AEDER whose telephone number is (571)272-8787. The examiner can normally be reached M-F 9am-6pm ET.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Samira Jean-Louis can be reached at (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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/SEAN E AEDER/Primary Examiner, Art Unit 1642
