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 .
Status of the Claims
Claim(s) 119-138 is/are pending and are under examination.
The following Office Action is in response to Applicant's communication dated 05/21/2026.
Restriction requirement from previous office actions is hereby withdrawn. Applicant’s argument
is found persuasive. In particular, applicant argues that the groups depend on an independent claim 119 and therefore i) requires every limitation of claim 119 and ii) the groups are not independent or distinct as claimed.
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.
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.
Claim(s) 119-138 is/are rejected under 35 U.S.C. 103 as being unpatentable over Frenz et al. US20200277664A1 in view of Christiansen et al. (US8335360B2)
Regarding claim 119, 121,123, 130, and 131 Frenz discloses method comprising: (a) contacting a section of a fixed biological sample with a nucleic acid stain; (e.g. sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. [¶0229]. H&E is a basic dye and used for staining acidic nuclear components (nucleic acids).)
(b) detecting an optical signal associated with the nucleic acid stain in the section of the fixed biological sample, wherein the optical signal associated with the nucleic acid stain is detected in the nucleus; (e.g. After staining nucleic acids, biological samples are imaged using various techniques such as fluorescence microscopy [¶0230], wherein biological sample includes cells that comprise nucleus or cellular macromolecules and organelles (e.g., mitochondria and nuclei) [¶0202-0209])
(c) comparing the optical signal detected to a reference to determine the quality of the fixed biological sample (e.g. the reference teaches comparing optical signal data to determine sample quality because it calculates a spatial RNA Integrity Number (sRIN), which is an in-situ indication of RNA quality based on an integrity score. Higher sRIN scores correlate with higher data quality. sRIN is generated using labeled cDNA image and a histology stain [¶1096])
However, Frenz does not disclose comparing optical signal of fixed sample to reference using quantitative ratio of signals detected in nucleus and cytoplasm.
Christiansen discloses automated calculation of nuclear to cytoplasmic stain ratio, AQUA® and C-AQUA analysis image analysis, wherein pixels are assigned to subcellular compartment is summed or averaged. The reference then validates image using “DAPI AQUA® score ratio (DAPI measured in nucleus/DAPI measured in cytoplasm) >1.5”. Hence, Christiansen teaches automated calculation of a ratio between nucleic acid stain signal detected in nucleus and signal detection.
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to incorporate a step of comparing optical signals of stained cells to a reference and analyze the sample quality using Christiansen’s automated nuclear to cytoplasmic ratio calculation into Frenz’s method. Frenz discloses staining fixed cell and detecting fluorescence signals to evaluate nucleic acid distribution. Frenz further discloses method to compares samples quality by calculating sRIN from labeled cDNA image and a histology stain. Christiansen teaches additional automated calculation of a nuclear to cytoplasmic signal ratio. Christiansen expressly state that the calculation improves detection of marker-defined cellular compartments [abstract]. Thus, one of ordinary sill in the art would have been motivated to apply Christiansen’s automated compartment-based ratio analysis to Frenz’s fixed cell nucleic acid staining method to provide more objective, reproducible, and high throughput comparison of nuclear and cytoplasmic stain signals, with reasonable expectation of success. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Regarding claim 120, Frenz further discloses method comprising de-crosslinking [¶0630].
Regarding claim 122, Frenz further discloses adjusting the level of fixation of an additional biological sample, wherein the additional biological sample is contacted with a nucleic acid probe that directly or indirectly binds to an analyte or a product thereof in the additional biological sample. (e.g. the reference discloses in some embodiments, tissue sample is fixed first time by freezing in liquid nitrogen. Next, tissue embedded in OCT, sectioned, and mounted on a spatial array with capture probes. then sample can be further chemically fixed and stained using a Hematoxylin and Eosin (H&E) staining protocol, which stains acidic cell structures (such as DNA and RNA in nuclei) [1322])
Regarding claim 124, Frenz further discloses the nucleic acid stain is a cyanine dye (e.g. SYTO Green [¶0179])
Regarding claim 125 , Frenz further discloses fixed biological sample is fixed using a fixing composition which optionally comprises 0.01-100% of fixative selected from the group consisting of: formaldehyde, glutaraldehyde, acetone, methanol, ethanol, acetic acid, potassium dichromate, chromic acid, potassium permanganate, B-5, Zenker's fixative, uranyl acetate, mercuric chloride, osmium tetroxide, and l-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC), picric acid, glyoxal, bis(sulfosuccinimidyl)suberate, and derivatives thereof. [¶0222-0224]
Regarding claim 126, Frenz further discloses the fixed biological sample is deparaffinized prior to staining (e.g. Prior to analysis, the paraffin-embedding material can be removed from the tissue section (e.g., deparaffinization) [¶0221]).
Regarding claim 127, Frenz further discloses the fixed biological sample is decrosslinked after staining and imaging (e.g. In some embodiments, formalin-fixation and paraffin-embedding stained samples can be treated with xylene to deparaffinize, then treated with de-crosslink methylene bridges, or both treatments can happen silutaneously. [¶1176])
Regarding claim 128, Frenz further discloses the fixed biological sample is additionally fixed wherein the additional fixation comprises contacting the fixed biological sample with a crosslinking agent. (e.g. In an example, a first fixation agent that is an organic solvent is provided to the hydrogel bead to change hydrogel bead volume and a second fixation agent that is a cross-linking agent is provided to the hydrogel bead to change hydrogel bead fluidity or rigidity. [¶0783])
Regarding claim 129, Frenz further discloses the fixed biological sample is contacted with an additional nucleic acid stain that binds to DNA or a cytoplasm stain (e.g.
Regarding claim 132, Frenz further discloses partitioning a cell in the additional portion of the fixed biological sample in a partition comprising a partition barcode. (e.g. the biological sample can be fixed on the substrate, the substrate then can be partitioned into two or more partitions containing beads, wherein the beads can have a plurality of spatial barcodes.[ ¶0617, 0623, and 0628])
Regarding claim 133, Frenz further discloses partition is an emulsion droplet or a microwell. (e.g. The partitions may be, for example, aqueous droplets included in a water-in-oil emulsion. The partitions may be, for example, a plurality of wells. [¶0643])
Regarding claim 134, Frenz further discloses sequencing a nucleic acid molecule or a portion thereof comprising .(i) a sequence of the nucleic acid probe or a complement thereof and .(ii) the partition barcode or a complement thereof.(e.g. the method further comprise sequencing to determine the sequence of the molecular tag barcode associated with the biological analyte bound to the capture probe and the spatial barcode of the capture probe [¶0016])
Regarding claim 135, Frenz further discloses the fixed biological sample is a formalin-fixed paraffin-embedded (FFPE) biological sample. [¶0220-0221]
Regarding claim 136, Frenz further discloses the analyte is an mRNA, and a first nucleic acid probe and a second nucleic acid probe are hybridized to a first analyte sequence and a second analyte sequence in the mRNA, respectively, wherein: the first nucleic acid probe comprises: .(i) a first hybridization region complementary to the first analyte sequence, and (ii) a first overhang; the second nucleic acid probe comprises: .(i) a second hybridization region complementary to the second analyte sequence, and .(ii) a second overhang; the first and second nucleic acid probes are ligated using the mRNA as template to form a ligated nucleic acid probe, with or without gap filling prior to the ligation; and the first and second overhangs independently comprise a primer binding sequence, a capture sequence, a barcode sequence, and/or a constant sequence. (e.g. In some aspects, the steps of RNA-templated ligation include: (1) hybridization of pairs of probes (e.g., DNA probes) to RNA within a tissue section; (2) ligation of adjacently annealed probe pairs in situ; (3) RNase H treatment that (i) releases RNA-templated ligation products from the tissue (4) amplification of RNA-templated ligation products (e.g., by multiplex PCR).[ ¶0933]. Probes comprises capture domain that hybridized with RNA.)
Regarding claim 137, Frenz further discloses transferring the ligated first and second nucleic acid probes to an array of features on a substrate, each of which comprises a spatial barcode sequence associated with a unique spatial location on the array and generating a nucleic acid molecule comprising (i) the spatial barcode sequence or a complement thereof and (ii) a sequence of the ligated first and second nucleic acid probes, or a complement thereof. (e.g. In some embodiments, a biological sample is embedded in a hydrogel to facilitate sample transfer to another location (e.g., to an array) on a substrate (e.g. glass slide) to perform spatial analysis. In some embodiments, a biological sample on a substrate can be covered with prepolymer solutions such that a hydrogel is formed around the biological sample. The biological sample contained in the hydrogel can then be contacted with a spatial array, and spatial analysis can be performed on the biological sample. [0241]).
Regarding claim 138, Frenz further discloses determining a sequence of the nucleic acid molecule (e.g. capture probes attached to array features can be released from the array features for sequencing, or remain attached to the array features, and the probes are sequenced while remaining attached to the array features (e.g., via in situ sequencing). [0412]).
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
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