DETAILED ACTION
This action is in response to Applicant’s response filed on 06/24/2026. The amendments to Claims 28, 31-32, and 36, filed on 06/24/2026, are entered. Claims 25-44 are now pending in the present application. This Action is made FINAL.
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 .
Response to Arguments
Applicant’s arguments filed on 06/24/2026 have been entered and fully considered.
Objections
With respect to the Objections to the Abstract and Claim 38, Applicant’s amendments to the Abstract and Claim 38 have overcome each and every objection previously set forth in the Office Action mailed on 02/24/2026. Accordingly, the objections to the Specifications and Claim 38 have been withdrawn.
35 U.S.C. 112 Rejections and Definiteness
With respect to the 112(a) and 112(b) rejections, Applicant’s arguments, see “35 U.S.C. 112 Rejections” and “Definiteness” sections on pages 7-9, filed on 06/24/2026, with respect to Claims 29, 31, 34, and 41-42 have been fully considered and are persuasive. Examiner acknowledges applicant has provided sufficient support found in the disclosure to overcome the 112(a) and 112(b) issues, including support found in at least paragraphs [0015], [0031], [0047], [0079]-[0080], [0083], [0103]-[0105], [0120], [0128], [0134]-[0135], and Figs. 4-6, 10a/b, 11a/b. Examiner further agrees with Applicant that the claims being broad does not render the claims unclear or indefinite, and the identified portions of the disclosure provided by the Applicant further render claims 29, 31, 34, and 41-42 definite. Accordingly, the 35 U.S.C. 112(a) and 35 U.S.C. 112(b) rejections of claims 29, 31, and 41-42, and the 35 U.S.C. 112(b) rejection of claim 34 have been withdrawn.
Art Rejections
With respect to the 35 U.S.C. 103 prior art rejections, Applicant's arguments, see “Art Rejections” section on pages 10-12, filed on 06/24/2026 have been fully considered but they are not persuasive.
Applicant argues that Dubois is directed to surface adherent platelet aggregates and therefore would not have suggested identifying platelet aggregates in the flowing sample of Hayedn. The argument is not persuasive. Although Dubois describes a surface adherent embodiment (e.g. Dubois ¶ [0028]), Dubois is not limited surface adherent analysis. Dubois claim 8 broadly recites quantitative analysis of blood aggregates using DHM, while dependent claim 9 specifically adds that the platelet aggregates are adhering on a surface. Dubois further expressly teaches that “using a flow chamber directly on the DHM” permits dynamic study of platelet aggregate formation in 3D (Dubois ¶ [0053]). Thus, Dubois expressly contemplates DHM analysis of platelet aggregates in a flow chamber and does not teach away from the proposed flow-based combination.
Hayden already teaches flowing biological entities, including platelets, through a microfluidic flow chamber and focusing the sample flow into a desired region aligned with the depth of field of the imaging device (Hayden ¶¶ [0023]-[0026], ¶ [0032], ¶ [0057]), including imaging using digital holographic microscopy (¶ [0087]). Accordingly, applying Dubois’s known DHM identification and analysis of platelet aggregates to Hayden’s focused, in-flow imaging arrangement would have been an obvious use of Dubois’s platelet aggregate analysis in the flow-based system of Hayden.
Applicant’s assertions regarding variations in aggregate size and possible flow-induced formation or disintegration are not persuasive. Hayden teaches focusing flow biological entities in the imaging region, and Dubois expressly teaches using a flow chamber with DHM to study platelet aggregates; refer to Hayden and Dubois paragraphs cited above. Thus, the cited references provide a reasonable basis for applying Dubois’s platelet aggregate analysis in Hayden’s flow-based system. Further claims 25 and 40 do not require avoiding aggregate formation or disintegration or determining the clinical indicators discussed by Applicant. Accordingly, Applicant’s arguments do not overcome the 35 U.S.C. 103 rejection.
Double Patenting
With respect to the Nonstatutory Double Patenting rejections of Claims 25, 28, 30, 37-38, 40, and 44, Applicant's arguments, see “Double Patenting” section on page 13, filed on 06/24/2026 have been fully considered but they are not persuasive.
Applicant argues that the provisional nonstatutory double patenting rejection is improper because the applied reference (US Application No. 18/291,943) claims include “MW requirements” not recited in the corresponding instant claims.
Respectfully, the argument is not persuasive. The “MW requirements” identified by Applicant, namely that the shear-thinning polymer have a molecular weight between 2 MDa and 10 MDa, constitute additional limitations that narrow the applied reference claims and do not, by themselves, render broader claimed subject matter patently distinct. See MPEP § 804, II.B.2, which explains that an examined claim may be unpatentable for nonstatutory double patenting where it is generic to a species or subgenus claimed in the conflicting application.
For clarity, reference claim 51 was previously relied upon for the Double Patenting rejection with respect to the limitations of instant claim 25, from which claims 28 and 37 depend. With respect to claim 28, reference claim 45 was relied upon for the additional limitation of determining a number of cells in the respective cell aggregate. With respect to claim 37, reference claim 44 was relied upon for the additional 2 MDa to 10 MDa molecular weight limitation. Thus, the rejection of claims 28 and 37 relies on reference claim 51 for the limitations inherited from claim 25 and reference claims 45 and 44, respectively, for the additional limitations of claims 28 and 37, as now reflected in the Double Rejection mapping found in the Double Rejection section below. The rejection is therefore maintained.
Upon further consideration and for clarity, reference claim 58 is no longer relied upon with respect to instant claim 25. The provisional nonstattutory double patenting rejection of claim 25 is maintained based on reference claims 44 and 51.
With respect to claim 38, Applicant’s argument regarding the MW requirement is not persuasive for the reasons discussed above. Nevertheless, upon reconsideration, the provisional nonstatutory double patenting rejection of claim 38 is withdrawn because the previously applied reference claims 44 and 50 do not require that single cells in the suspension are also focused in the focal plane, as required by the instant claim 38.
With respect to the Nonstatutory Double Patenting rejections of Claim 32, Applicant's arguments, see “Double Patenting” section on page 13, filed on 06/24/2026 have been fully considered and are persuasive. The nonstatutory Double Patenting rejection of Claim 32 has been withdrawn.
Accordingly, the provisional nonstatutory double patenting rejection of claims 32 and 38 is withdrawn and Applicant’s arguments are otherwise not persuasive. The provisional nonstatutory double patenting rejection of claims 25, 28, 30, 37, 40, and 44 are maintained.
See maintained Double Patenting and 35 U.S.C. 103 rejections below for further details.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 25, 28, 30, 37, 40, and 44 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims of copending Application No. 18/291,943 (reference application).
Although the claims at issue are not identical, the examined claims are not patentably distinct from the corresponding claims of the reference application because they are broader than, encompass, or constitute obvious variations of the subject matter defined by the reference claims, as set forth below. See the mapping of the Instant claims to the corresponding Reference claims found in copending Application No. 18/291,943 in the table below.
Instant claims
Reference claims
25
44 + 51
28
45 + 51
30
51
37
44 + 51
40
54
44
54 + 55
With respect to claim 28, applying the cell number determination of reference claim 45 to the platelet aggregates recited by reference claim 51 would have been an obvious variation of the cell aggregate detection method of the reference application.
With respect to claims 40 and 44, reference claims 54 and 55 broadly recite biological cells and cell aggregates, while the supporting disclosure expressly describes whole blood or blood-fraction samples containing platelet aggregates and identification of platelet aggregates (see ¶ [0042] and ¶ [0047] of the reference application’s pre-grant publication, US 20240377310 A1). Accordingly, limiting the devices of reference claims 54 and 55 to expressly disclosed blood/platelet aggregate embodiment would have been an obvious variation.
The remaining mapped claims recite the same subject matter or broader subject matter encompassing the corresponding reference claims, as shown in the table above.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
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.
For examination purposes, please refer to 35 U.S.C. 112(b) issues identified above and the interpretation of “predefined number” and “cells of any type”. The corresponding interpretations of the indefinite terms or phrases are applied throughout the section found below.
Claims 25-27 are rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1).
Regarding Claim 25,
Hayden et al. teaches: a method for detecting (Hayden et al. teaches a method for aligning a biological entity (see ¶ [0024] “method for aligning a non-spherical biological entity carried in a sample into a desired region in a flow cell”), including biological cells (¶ [0054] “..i.e., one or more non-spherical biological entities such as red blood cells..”), using an imaging device (see ¶ [0002]) that detects interference patterns representing red blood cell (RBC) characteristics, structures, etc. (¶ [0083] “imaging device 90 includes an interferometry unit (not shown) and a detector…object information is filtered out or deleted from the reference beam and then the filtered reference beam is superimposed with the object beam to detect the interference pattern at the detector…The interference pattern also referred to as image of the RBC 4 represents characteristics of the RBC 4 such as physical structures in the RBC 4, morphology of the RBC 4, and so on and so forth.”) using a quantitative phase-contrast microscope (¶ [0087] “the present technique …using digital holographic microscopy device”; Examiner notes ¶ [0013] of the instant application’s disclosure states, “The quantitative phase-contrast microscope may for example be a ptychographic imaging device or a digital holographic microscope…”), the method comprising:
preparing a suspension comprising biological cells from a sample (See suspension process described in ¶ [0024], including “aligning a non-spherical biological entity carried in a sample into a desired region in a flow cell.”);
generating a flow of the suspension along a microfluidic channel to one or both of viscoelastically and hydrodynamically focus cell (See ¶¶ [0023]- [0026] “…the flow chamber is a microfluidic channel…The flow cell includes a flow chamber having a rectangular cross-section, a top wall, a bottom wall opposite to the top wall, a first side wall, a second side wall opposite to the first side wall and the desired region. In the method, a first viscoelastic fluid, hereinafter also referred to as the first fluid, is provided to the flow chamber such that the first fluid laminarly flows in the flow chamber in the form of a bottom laminar flow along the bottom wall from one end of the flow chamber towards another end of the flow chamber…the desired region is aligned with the depth of field in the field of view of the imaging device”. The “desired region” in the “field of view” of the “flow chamber” taught in Hayden et al. is interpreted to be equivalent to the “suspension in a focal plane of the quantitative phase-contrast microscope” claimed in the instant application.)
taking one or more phase (¶ [0057] “when the object is positioned in the depth of field around the focus of the imaging device 90, an ‘in-focus’ image of the object is obtainable…The region within the depth of field around the focus of the imaging device 90 is a region (not shown) in which the object should be ideally positioned or focused or concentrated within the flow chamber 10 for obtaining in-focus images or interference patterns of the object”; ¶ [0087] “By using the present technique and applying it to image RBCs 4 using digital holographic microscopy device 90, holograms, i.e., phase image and bright field image is recorded with a velocity 50-200 frames per second.”; Examiner notes, Hayden’s teaching of a digital holographic microscopy device (¶ [0087]) is a known quantitative phase contrast imaging technique and therefore corresponds to the claimed quantitative phase contrast microscope.); and
identifying cell (The previous limitation details interference patterns ¶ [0057] are found in phase images of red blood cells (RBC), taught in ¶ [0087]. Hayden et al. also teaches interference pattern includes RBC characteristics such as physical structures, morphology, etc. and the imaging device detects and analyzes the interference patterns (refer to ¶ [0083]). Examiner interprets identifying cells in the one or more phase images to be equivalent to detecting and analyzing red blood cells found in phase images of interference patterns.),
wherein the sample is a whole blood sample or a blood fraction sample (¶ [0054] “…the sample with its components, i.e., one or more non-spherical biological entities such as red blood cells…”); and identifying cell (In addition to identifying cells in the one or more phase shift images, taught in prior limitations, Hayden et al. teaches [0032] “The non-spherical biological entity may be, but not limited to, an erythrocyte, a platelet, an irregularly shaped leukocyte, and so on and so forth.”.
Hayden et al. fails to teach detecting or identifying cell aggregates, including platelet aggregates, and while Hayden et al. teaches phase images, Hayden et al. fails to explicitly teach phase shift images.
In a related art, Dubois et al. teaches: a method for quantitative analysis of blood cells, platelets, or aggregates of cells and platelets using digital holographic microscopy (Abstract; ¶ [0010]) and quantitative phase contrast imaging (see ¶ [0025]). Dubois et al. further teaches detecting (see Dubois et al. ¶ [0034] “the recorded fields of view by the DHM show aggregates”; ¶ [0042] “the regions covered by the aggregates were detected.”), determining aggregate information from a corresponding image (¶ [0034] “…measure the aggregate shapes thanks to the quantitative phase contrast imaging capability of the DHM.”; ¶ [0046] “The volume of the aggregate…is obtained by computing in the corresponding phase image”). Dubois et al. also expressly teaches that “…using a flow chamber directly on the DHM” platelet aggregate formation may be dynamically studied (¶ [0053]). See also claim 8, which recites DHM analysis of blood platelet aggregates, and dependent claim 9, which separately limits the platelet aggregates to those adhering on a surface. The phase images used by Dubois et al. represent optical phase information obtained by quantitative phase contrast DHM and therefore correspond to the claimed phase shift images.
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to modify Hayden et al.’s focused, flow-based DHM imaging method to identify platelet aggregates in the phase images using the platelet-aggregate detection and analysis taught by Dubois et al. Hayden et al. provides an arrangement for focusing biological entities, including platelets, within the imaging region of a flow chamber, while Dubois et al. teaches DHM identification and analysis of platelet aggregates and expressly contemplates performing such analysis using a flow chamber directly on the DHM (Dubois ¶ [0053]). A person of ordinary skill in the art therefore would have had reason to apply Dubois et al.’s platelet aggregate analysis to Hayden et al.’s in-flow imaging arrangement to permit platelet aggregates carried in the sample flow to be positioned in the imaging region and identified from the resulting phase images.
Regarding Claim 26,
Hayden et al. and Dubois et al. teach the limitations of claim 25.
Dubois et al. further teaches: determining a total number or fraction of platelet aggregates in the one or more phase shift images (Dubois et al. previously taught (found in claim 25 above) the use of phase shift images for determining aggregates. Dubois et al. further teaches an example of a platelet aggregate (i.e. “platelet aggregates formation”) for determining a total number of platelet aggregates in the image, ¶ [0028] “The platelet aggregates formation… images on a circumferential plane from the wells were captured by the image analyzer…which quantifies the platelet aggregates formed…the results were expressed as number of aggregates detected”. Because the aggregates are detected using reconstructed quantitative phase images obtained by digital holographic microscopy, the platelet aggregate count is determined from one or more phase shift images.).
Regarding Claim 27,
Hayden et al. and Dubois et al. teach the limitations of claim 26.
Dubois et al. further teaches: using the total number or fraction of platelet aggregates as an indicator for complications of an infected patient (Dubois et al. teaches COPD (chronic obstructive pulmonary disease) as an example case for the detection method (refer to ¶¶ [0054]- [0056]), and ¶ [0056] states “The present example shows the interest of the quantitative data that can be obtained by the method of the invention in helping diagnosis of COPD complications.” The numerical data of platelet aggregates taught in claim 26 (refer back to ¶ [0028]) constitutes quantitative data. In the COPD case example, the application teaches that quantitative data obtained by the method of the invention is used as an indicator to help diagnose complications associated with COPD. Accordingly, it would have been understood that the total number of platelet aggregates taught by Dubois et al. constitutes quantitative data that may be used as an indicator for complications of an infected patient.).
Claims 28-29 are rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), and in further view of Dimarzio & Warger (US 20080032325 A1).
Regarding Claim 28,
Hayden et al. and Dubois et al. teach the limitations of claim 25.
Dubois et al. further to teaches: wherein identifying cell aggregates in the one or more phase shift images (Refer to ¶ [0034], ¶ [0042], ¶ [0046] and claim 25 above for further detail)
Hayden et al. and Dubois et al. fails to teach: determining a number of cells in the respective cell aggregate.
In a related art, DiMarzio & Warger teach: wherein identifying the cell aggregates in the one or more phase shift images comprises determining a number of cells in the respective cell aggregate (DiMarzio & Warger specifically teaches a method and a device for obtaining a full field phasing image technique that is an optical path length deviation (OPD) image (i.e. phase-shift representation or images) identifying individual cells and cell clusters (i.e. cell aggregates), and determining the number of cells in the respective cell aggregate by subtracting cells from the OPD image repeatedly until no cells are left in the cell cluster (Abstract; ¶ [0013]); Abstract “The cell count is obtained from the number of cells subtracted.”).
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a method that identifies cell aggregates in one or more phase shift images to also determine the number of cells in the respective aggregate as taught by DiMarzio & Warger. Doing so would increase the amount of quantitative phase-contrast information to the method. The inventions lie in the same field of endeavor of determining quantitative cell data from microscopic image devices. Although one reference (DiMarzio & Warger) applies the techniques to embryos and the other (Hayden et al. and Dubois et al.) to blood and platelet cells, both address the same technical field of quantitative microscopic image analysis of biological cells, and differ only in the type of biological specimen analyzed. Further motivation to combine the teachings include, “a need for an instrument that can non-invasively count the number of cells… in order to help physicians make better determinations...” (DiMarzio & Warger [0005]).
Regarding Claim 29,
Hayden et al. and Dubois et al. teach the limitations of claim 25.
Dubois et al. further teaches: determining a number or fraction of platelet aggregates (see Debois et al. ¶ [0028])
Hayden et al. and Dubois et al. fail to teach a predefined number of cells for determining a number or fraction of platelet aggregates. Examiner interprets “predefined number” to be equivalent to a “threshold”.
In a related art, DiMarzio & Warger teaches: a preset threshold value of residual phase representing further cells (see ¶¶ [0012]- [0013]). DiMarzio & Warger uses this threshold when subtracting and counting cells detected in a cluster or aggregate. The preset threshold can be set either manually by a user or automatically by the program, and the value is a number corresponding to a volume corresponding to a cell, cell fragment, or polar body (¶¶ [105]- [0106]).
It would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a method that determines a number or fraction of platelet aggregates to also include a threshold or predefined number of cells framework, as taught by DiMarzio & Warger, to determines what aggregates are quantitively analyzed. The inventions lie in the same field of endeavor of determining quantitative cell data from microscopic image devices. Although one reference (DiMarzio & Warger) applies the techniques to embryos and the other (Hayden et al. and Dubois et al.) to blood and platelet cells, both address the same technical field of quantitative microscopic image analysis of biological cells, and differ only in the type of biological specimen analyzed. Combining the reference’s teachings would increase the accuracy of the phase-contrast method relied on in helping physicians make better determinations (DiMarzio & Warger ¶ [0005]) by providing additional parameters (e.g. predefined number or cell thresholds) and eliminate unnecessary results (e.g. number of cells not matching predetermined value or data below the threshold) and excessive time associated with performing these unnecessary tasks.
Claim 30 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), and in further view of Mahan & Stewart (AU 2002233923 A1).
Regarding Claim 30,
Hayden et al. and Dubois et al. teach the limitations of claim 25.
Hayden et al. further teaches: wherein identifying cell (Hayden et al. teaches identifying cells in the one or more phase images, as previously detailed in claim 25 (found above in this section) and in Hayden et al. ¶ [0057], ¶ [0087], ¶ [0083].)
Hayden et al. fails to teach identifying cell aggregates and leukocyte-platelet aggregates in phase shift images.
In a related art, Duboi et al. teaches: a method of quantitative analysis of blood cells, platelets, or aggregates of cells and platelets using digital holographic microscopy (Abstract; ¶ [0010]) and quantitative phase contrast imaging (see ¶ [0025]). Duboi et al. further teaches determining/identifying aggregates (see Duboi et al. ¶ [0034]; ¶ [0042]), performing this task using phase shift imaging (¶ [0034]; ¶ [0046]; The phase image used to identify aggregates represents phase shifts because the identification of aggregates depends on detecting regions exhibiting increased optical phase delay relative to surrounding regions. As a result, the phase image inherently functions as a phase shift image, even if not expressly labeled as such.), and the use of a flow chamber in the digital holographic microscopy allows for study of platelet aggregates (¶ [0053]).
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al.’s teachings of a method that identifies cells using phase images to account for aggregates of biological cells and platelets by using phase shift images, as taught by Dubois et al. The inventions lie in the same field of endeavor of detecting blood cells and platelets using a digital holographic microscopy. The motivation to combine the references is to make the detection system more robust and accurate by accounting for more parameters (e.g. aggregates). Furthermore, the flow chamber’s implementation in relation to cell or platelet aggregates (taught by Dubois) would address a need of aligning non-spherical biological entities in a desired region (Hayden et al. ¶ [0006]).
Hayden et al. and Duboi et al. fail to teach identifying leukocyte-platelet aggregates.
In a related art, Mahan & Stewart teach: platelet-leukocyte interaction, i.e., formation of leukocyte-platelet aggregates is found in the sample (Refer to Mahan & Stewart p. 6, lines 8-10, “The present invention relates to a platelet/leukocyte interaction assay… the present invention monitors interaction between platelets and leukocytes in the blood or blood-derived sample.”), the interaction is treated as an identifiable cellular complex (p. 7, lines 23-26, “Evaluation of the interaction between the platelets and leukocytes comprises attachment of the cells to the solid-phase support… such that either qualitative and/or quantitative analysis of the interaction can be accomplished.”), the platelet-leukocyte complexes are detected (p. 8, lines 4-6, “Such analysis can be performed using any method capable of detecting and counting the number of platelet/leukocyte/solid-phase support complexes present in the assayed sample.”), and leukocyte-platelet complexes are identified as aggregates (p. 18, lines 11-12, “Phase contrast microscopy confirmed leukocyte/platelet complex association… in aggregates”, and the leuokocyte-platelet complexes), thus the platelet-leukocyte complexes detected and identified are equivalent to leukocyte-platelet aggregates.
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to apply the aggregate identification techniques taught by Dubois et al., which operate on phase shift images, to the phase-based cell identification framework taught by Hayden et al., and identify leukocyte-platelet aggregates as taught by Mahan & Stewart, since platelet adhesion to leukocytes was known to form identifiable aggregates of interest. Accordingly, the combination teaches or renders obvious identifying leukocyte-platelet aggregates in the one or more phase shift images. The inventions lie in the same field of endeavor of microscopy-based identification and analysis of biological cells. Although the references analyze different types of biological cells, they address the same technological problems using similar techniques. Mahan & Stewart teach platelet leukocyte interactions are monitored in blood samples (Mahan & Stewart p. 6, lines 8-10), indicating leukocyte-platelet aggregates are a known and relevant target for analysis. The motivation to combine the teachings would have been to apply known phase shift image-based aggregate identification techniques to identify leukocyte-platelet aggregates, resulting in a more robust framework and increased data available to users (i.e. physicians) for studying and diagnosing diseases.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of Mahan & Stewart (AU 2002233923 A1), and in further view of DiMarzio & Warger (US 20080032325 A1),
Regarding Claim 31,
Hayden et al., Dubois et al., and Mahan & Stewart teach the limitations of claim 30.
Hayden et al. and Dubois et al. further teach: determining a total number or fraction of (mirrors limitation in Claim 26 taught by Hayden et al. and Dubois et al.). This limitation equally mirrors the limitation found in claim 26. For the sake of brevity, please refer to claim 26 for Hayden et al. and Dubois et al. teachings of the corresponding limitation. The teachings in claim 26 and the motivations to combine found in claim 25 apply here.
The method taught by Hayden et al., Dubois et al., and Mahan & Stewart fails to explicitly disclose teach: leukocyte platelet and determining a number or fraction of leukocyte-platelet aggregates comprising at least a predefined number of cells
However, Hayden et al., Dubois et al., and DiMarzio & Warger teach: determining a number or fraction of (mirrors limitation in Claim 29 taught by Hayden et al., Dubois et al., and DiMarzio & Warger). This limitation equally mirrors the limitations found in claims 29. For the sake of brevity, please refer to claim 29 for Hayden et al., Dubois et al., and DiMarzio & Warger’s teachings of the corresponding limitation. The motivation to combine the teachings of Hayden et al., Dubois et al., and DiMarzio & Warger for the limitations of claim 31 is equivalent to the motivation to combine the respective references detailed in claim 29.
The method of Hayden et al., Dubois et al., Mahan & Stewart, and DiMarzio & Warger fail to specifically teach addressing leukocyte-platelet aggregates when determining a total number or fraction of aggregates in the one or more phase shift images.
However, Mahan & Stewart further teach: leukocyte-platelet aggregates by showing platelet-leukocyte interaction, i.e., formation of leukocyte-platelet aggregates is found in the sample (Refer to Mahan & Stewart p. 6, lines 8-10), the interaction is treated as an identifiable cellular complex (p. 7, lines 23-26), the platelet-leukocyte complexes are detected (p. 8, lines 4-6), and leukocyte-platelet complexes are identified as aggregates (p. 18, lines 11-12), thus the platelet-leukocyte complexes detected and identified are equivalent to leukocyte-platelet aggregates. Further, Mahan & Stewart teaches counting platelet-leukocyte complexes (i.e. leukocyte-platelet aggregates) (see p. 8, lines 3-6 “The assay can also be used for quantitative or semi-quantitative determination of the platelet-leukocyte interaction. Such analysis can be performed using any method capable of detecting and counting the number of platelet/leukocyte/solid-phase support complexes present in the assayed sample.”)
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to apply techniques of determining the number or fraction of platelet aggregates taught by Dubois et al., which operate on phase shift images, to also account for determining the number of leukocyte-platelet aggregates through the use of platelet-leukocyte complexes taught by Mahan & Stewart. One of ordinary skill in the art at the time of the invention would also be able to determine a number or fraction of leukocyte-platelet aggregates (using the teachings from Dubois et al. and Mahan & Stewart) using a predefined threshold of cells, taught by DiMarzio & Warger, to determine if the leukocyte-platelet aggregate is to be counted. Accordingly, the combination teaches or renders obvious determination of total number or fraction of leukocyte-platelet aggregates in the one or more phase shift images and based on predefined number of cell conditions. The inventions lie in the same field of endeavor of microscopy-based identification and analysis of biological cells. Although the references analyze different types of biological cells, they address the same technological problems using similar techniques. Mahan & Stewart teach platelet leukocyte interactions are monitored in blood samples (Mahan & Stewart p. 6, lines 8-10), indicating leukocyte-platelet aggregates are a known and relevant target for analysis. The motivation to combine the teachings would have been to apply known phase shift image-based aggregate identification and quantitative techniques to quantitively analyze leukocyte-platelet aggregates, resulting in a more robust framework and increased data available to users (i.e. physicians) for studying and diagnosing diseases.
Claims 32 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of Mahan & Stewart (AU 2002233923 A1), and in further view of Michelson et al. ("Circulating Monocyte-Platelet Aggregates Are a More Sensitive Marker of In Vivo Platelet Activation Than Platelet Surface P-Selectin: Studies in Baboons, Human Coronary Intervention, and Human Acute Myocardial Infarction", 2001; Copy was provided by applicant in the instant application and IDS).
Regarding Claim 32,
Hayden et al., Dubois et al., and Mahan & Stewart teach the limitations of claim 30.
Hayden et al., Dubois et al., and Mahan & Stewart fail to explicitly teach: determining a number or fraction of leukocyte-platelet aggregates comprising at least a predefined number of leukocytes.
In a related art, Michelson et al. teaches: determining a number or fraction of leukocyte-platelet aggregates comprising at least a predefined number of leukocytes (Michaelson et al. teaches identifying monocyte-platelet and neutrophil-platelet aggregates, which are leukocyte-platelet aggregates (p. 1535, left column, sub-section Leukocyte-Platelet Aggregates “the thrombin-activated infused (biotinylated) platelets formed circulating monocyte-platelet and neutrophil-platelet aggregates”; p. 1534, left column, sub-section Leukocyte-Platelet Aggregates “Circulating neutrophils and monocytes with adherent infused platelets were identified by positivity for both RED670 … (i.e. the infused biotinylated platelets) and FITC with … (i.e. the leukocyte binding of the platelet-specific anti--GP IIa monoclonal antibody Y2/51).”). Michaelson et al. further teaches determining a number or fraction of leukocyte platelet aggregates, stating that “Data are expressed as a percentage of all neutrophils or monocytes positive for infused platelets.” (p. 1534, left column, sub-section Leukocyte-Platelet Aggregates) and “Monocyte-platelet and neutrophil-platelet aggregates are expressed as percentage of all monocytes and neutrophils with adherent platelets.” (p. 1536, Fig. 3 description). Michaelson et al. also teaches predefined gating thresholds for flow analysis. Specifically, positivity is defined using preset fluorescence thresholds (p. 1535, left column, sub-section Platelet Surface P-Selection “The percentage of P selectin-positive platelets was defined as the percentage of platelets that had a FITC or PE fluorescence greater than a threshold determined by 99% of platelets incubated with purified FJTC- or PE-conjugated mouse lgG isotypic controls.”). Thus, event classification requires predefined fluorescence thresholds are met prior to analysis. In summary, leukocyte-platelet aggregates are defined as leukocyte-positive events (monocytes or neutrophils identified by CD14 staining and light scatter characteristics) that also meet predefined fluorescence criteria for platelet association, each identified aggregate necessarily comprises at least a predefined number of leukocytes (i.e., at least leukocyte satisfying preset gating thresholds). Therefore, Michelson et al. discloses determining a number or fraction of leukocyte-platelet aggregates comprising at least a predefined number of leukocytes.).
It would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified the method for detecting cell aggregates of biological cells using a quantitative-contrast microscope taught by Hayden et al., and previously modified by Dubois et al. and Mahan & Stewart, to incorporate the teachings of Michelson et al. because both references lie in the same field of endeavor of quantitative biological cell and/or aggregate analysis and Michelson et al.’s teachings would increase the amount of quantitative data retrieved in Hayden et al., Dubois et al., and Mahan & Stewart’s teachings by also determining a number or fraction of leukocyte-platelet aggregates comprising at least a predefined number of leukocytes as taught by Michelson et al.
Claims 33 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of Mahan & Stewart (AU 2002233923 A1), in further view of Michelson et al. ("Circulating Monocyte-Platelet Aggregates Are a More Sensitive Marker of In Vivo Platelet Activation Than Platelet Surface P-Selectin: Studies in Baboons, Human Coronary Intervention, and Human Acute Myocardial Infarction", 2001; Copy was provided by applicant in the instant application and IDS), and in further view of Rich (US 20130091937 A1).
Regarding Claim 33,
Hayden et al., Dubois et al., Mahan & Stewart, and Michelson et al. teach the limitations of claim 32,
Michelson et al. further teaches: the method comprises one or both of determining a number or fraction of leukocyte-platelet aggregates comprising
one or both of the presence of leukocyte-platelet aggregates comprising These limitations are taught by Michelson et al. in claim 32. The teachings and motivation to combine in claim 32 apply here.
Hayden et al., Dubois et al., Mahan & Stewart, and Michelson et al. fail to explicitly teach determining leukocyte-platelet aggregates based on a threshold of two or more leukocytes or three or more leukocytes present in the leukocyte-platelet aggregate, and using the presence of two or more leukocytes or three or more leukocytes as an indicator for an infection.
However, Dubois et al. further teaches: aggregates used as an indicator for an infection (Dubois et al. teaches COPD (chronic obstructive pulmonary disease) as an example case for the detection method (refer to ¶¶ [0054]- [0056]), and ¶ [0056] states “The present example shows the interest of the quantitative data that can be obtained by the method of the invention in helping diagnosis of COPD complications.” The numerical data of platelet aggregates taught in claim 26 (refer back to ¶ [0028]) constitutes quantitative data. In the COPD case example, the application teaches that quantitative data obtained by the method of the invention is used as an indicator to help diagnose complications associated with COPD. Accordingly, it would have been understood that the total number of platelet aggregates taught by Dubois et al. constitutes quantitative data that may be used as an indicator for complications of an infected patient.).
It would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified the quantitative leukocyte and leukocyte-platelet aggregate analysis taught by Hayden et al., Dubois et al., Mahan & Stewart, and Michelson et al. to incorporate an indicator for an infection, as taught by Dubois et al., based on the results of the analysis. Doing so would provide aid users (e.g. physicians) in diagnosing patients. These inventions lie in the same field of endeavor of quantitative biological cell and aggregate analysis.
Hayden et al., Dubois et al., Mahan & Stewart, and Michelson et al. fail to explicitly teach leukocyte thresholds of two or more, and three or more for determining a number or fraction of leukocyte-platelet aggregates and as an indicator for infection.
In a related art, Rich teaches: a fluidic system that recognizes aggregates of particle events found in the data set that automatically adjusts the flow rate of the sample fluid when aggregate particle event in which particles in the sample fluid closely spaced flow through the interrogation zone (Abstract; ¶ [0026] “analysis engine recognizes an aggregate particle event in which two or more particles in the sample fluid closely spaced to each other have passed through the interrogation zone”). Rich further teaches a threshold or predetermined number of particles may be set at different numbers (¶ [0030] “after the analysis engine has recognized a predetermined number of aggregate particle events (e.g., one, two, or five)”), and when the threshold is met, an action is triggered (¶ [0030] “the controller may automatically adjust the flow rate of the sample fluid and/or core stream diameter after the analysis engine has recognized a certain threshold percentage of aggregate particles”).
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al., previously modified by Dubois et al., Mahan and Stewart, and Michelson et al.’s teachings of leukocyte-platelet aggregate identification and quantification methodologies to incorporate Rich’s aggregate particle event detection and predetermined threshold logic in order to further characterize leukocyte-platelet aggregates based on the number of present leukocytes because leukocytes are particles/cells suspended in fluid and because aggregate detection and threshold-based classification techniques were well established. Applying Rich’s methodology to leukocyte events would have been a predictable use of known analytical techniques, yielding identification and quantification of leukocyte-platelet aggregates comprising at least a predefined number (e.g. two or more, three or more) of leukocytes, which in turn could be used as an indicator of infection (as taught by Dubois et al.). Hayden et al., Dubois et al., Michelson et al., and Rich all teach identifying and quantifying fluid samples using flow-based detection. Accordingly, the references all address flow-based particle, event detection, and aggregate classification techniques. The motivation to combine references would be to identify specific aggregates more quickly by using parameters (i.e. number of particles or leukocytes and predetermined thresholds) to identify aggregates and help physician’s identify infections more accurately and rapidly.
Claims 34 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of Michelson et al. ("Circulating Monocyte-Platelet Aggregates Are a More Sensitive Marker of In Vivo Platelet Activation Than Platelet Surface P-Selectin: Studies in Baboons, Human Coronary Intervention, and Human Acute Myocardial Infarction", 2001; Copy was provided by applicant in the instant application and IDS).
Regarding Claim 34,
Hayden et al. and Dubois et al. teach the limitations of claim 25.
Dubois et al. further teaches: determining a total number or fraction of aggregated (Dubois et al. teaches acquiring quantitative phase (phase-shift) images of a biological samples and identifying aggregates within such images, including by determining a number of aggregates formed (see Abstract; ¶[0025] “The present invention discloses a method able to characterize the 3D platelets or aggregates shapes by using the quantitative phase contrast imaging provided by DHM or DDHM.; ¶ [0022] FIG. 3 “Phase image showing platelet aggregates obtained with DHM”; ¶ [0028] “image analyzer… quantifies the platelet aggregates formed... The results were expressed as number of aggregates detected…”)
Hayden et al. and Dubois et al. fail to explicitly disclose platelet containing mixed cell aggregates.
In a related art, Michelson et al. teaches: that platelets form mixed cell aggregates, including monocyte-platelet and neutrophil-platelet aggregates (p. 1535, left column, sub-section Leukocyte-Platelet Aggregates and p. 1534, left column, sub-section Leukocyte-Platelet Aggregates), and determines a fraction of aggregates within the sample (p. 1534, left column, sub-section Leukocyte-Platelet Aggregates; p. 1536, Fig. 3 description). Monocyte-platelet and neutrophil-platelet aggregates are leukocyte-platelet aggregates and deemed a type of cell aggregate containing aggregated platelets.
It would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to apply the known method identifying platelet containing mixed cell aggregates taught by Michelson et al. to the phase-shift imaging aggregate analysis taught by Hayden et al., previously modified by Dubois et al., in order to quantify aggregated platelets contained within cell aggregates of any type identified in one or more phase shift images. Hayden et al., Dubois et al., and Michelson et al. lie in the same field of endeavor of analyzing cells in biological samples, while Michelson et al. and Dubois et al. both focus on analyzing aggregates in biological samples and quantify aggregates. Their combination would have yielded the predictable result of determining a total number or fraction of aggregated platelets contained in cell aggregates of any type found in a phase shift image. The motivation to combine would be to improve quantitative analysis results in cellular biology.
Claims 35 and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of Holmes et al. (US 20140038206 A1).
Regarding Claim 35,
Hayden et al. and Dubois et al. teach the limitations of claim 25.
Hayden et al. and Dubois et al. fail to teach: determining one or more of a granularity measure, a size distribution of a plurality of cells and one or more parameters pertaining to said size distribution from the one or more phase shift images, wherein the granularity measure characterizes a granularity of one or more cells in the one or more phase shift images.
In a related art, Holmes et al. teach: determining one or more of a granularity measure, a size distribution of a plurality of cells (Holmes et al. teaches quantitative microscopy includes statistical image processing (¶ [0062] “Quantitative microscopy may include use of image analysis techniques and/or statistical learning and classification methods to process images obtained by microscopy.”) and cell size and volume are cellular attributes (¶ [0064]; ¶ [0065]). Holmes et al. further teaches measuring more than one cells and recognizing size, granularity and size distribution of cells (¶ [0141] “…image of cells prepared and analyzed… may include no cells, one cell, or multiple cells.”; ¶ [0142] “the assay system is configured to perform cytometry assays. Cytometry assays are typically used to optically, electrically, or acoustically measure characteristics of individual cells…Such characteristics include but are not limited to size; shape; granularity”) and one or more parameters pertaining to said size distribution from the one or more phase shift images (¶ [0147] “The intensity of fluorescence distribution for individual platelets was measured…a fit for the intensity distribution was determined…Parameters of the fit, such as the mean of the Gaussian, the variance, the volume, the width, and the area of the base, etc.”; In addition, Holmes et al. teaches determining a distribution of red blood cell width (RDW) in ¶ [0165], which is another parameter of a cell size distribution derived from measured cell sizes.), wherein the granularity measure characterizes a granularity of one or more cells in the one or more phase shift images (More than one cells are being measured (¶ [0141]); Holmes et al. teaches the quantitative microscopy methods use phase shift imaging to collect the attributes described above (¶ [0062] “methods, systems, and devices are provided herein for quantitative microscopy. Quantitative microscopy may involve… quantitative phase contrast microscopy methods to measure one or more cellular attributes…”)). Accordingly, Holmes et al. teaches determining a size distribution of a plurality of cells and at least one parameter pertaining to the size distribution from phase shift images, as recited in the claim. Additionally, Holmes et al. teaches determining granularity across one or more cells from the phase shift images, as recited in the claim.
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a method of quantitative analysis of biological cells to incorporated Holmes et al.’s known teachings of determining sizes of cells and a size distribution or granularity of a plurality of cells and at least one parameter, including a mean of the Gaussian, a variance, a volume, a width, or an area of the base of cells, using phase shift images. Doing so would increase the amount of data derived from a quantitative biological cell method and improve a user’s (e.g. physician) basis for analysis, diagnosis, etc. The inventions lie in the same field of endeavor of quantitative biological cell analysis using microscopes.
Regarding Claim 36,
Hayden et al., Dubois et al., and Holmes et al. teach the limitations of claim 35.
Holmes et al. further teaches: wherein one or more of the granularity measure, the size distribution and the one or more parameters pertaining to said size distribution are determined for one or both of single and aggregated cells of a particular type. Holmes et al. teaches determining cell attributes from quantitative phase contrast microscopy (¶ [0062] quantitative phase contrast microscopy methods to measure one or more cellular attributes…”) and that quantitative microscopy include image analysis and statistical learning and classification obtained by microscopy (¶ [0062]). Holmes et al. goes on to expressly teach the measurable attributes include cell size, granularity, volume, shape, and area (¶ [0064]; ¶ [0065]; ¶ [0142]), thereby teaching determination of size and other attributes from phase images. Holmes et al. further teaches an image may include multiple cells (¶ [0141]) and defines cells to include individual cells and “small groups of cells” (¶ [0142]), thereby expressly including both cells and aggregated (grouped) cells. Holmes et al. also recognizes size distribution and determining different hematologic parameters such mean, variance, volume, area of the base of the cell, and specifically a red blood cell distribution width (RDW) (¶ [0147]; ¶ [0165]), which is a parameter pertaining to a size distribution for a particular cell type (red blood cells). In summary, Holmes et al. teaches determining a size distribution, granularity, and at least one parameter pertaining to that size distribution for both single and aggregated cells of a particular type (e.g. red blood cells), as recited in the claim.
Claim 37 and 39 are rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), and in further view of Kwon et al. (“Particle Focusing under Newtonian and Viscoelastic Flow in a Straight Rhombic Microchannel”, 2020; Copy provided by examiner with Office Action mailed on 02/24/2026).
Regarding Claim 37,
Hayden et al. and Dubois et al. teach the limitations of claim 25, including a suspension comprising a viscoelastic fluid.
Hayden et al. further teaches: the use of shear-thinning polymer and a polymer concentration including 0.1%, which is less than 0.2% as claimed in the instant claim 37 (¶ [0079]).
Hayden et al. fails to teach the shear-thinning polymer having a molecular weight between 2 MDa and 10 MDa.
In a related art, Kwon et al. teaches: viscoelastic fluid comprises a shear-thinning polymer having a molecular weight between 2 MDa and 10 MDa and wherein a mass fraction of the shear-thinning polymer in the suspension is less than 0.2% (Kwon et al. teaches viscoelastic fluid focusing using poly(ethylene oxide) (PEO) with a molecular weight of approximately 2 MDa and polymer concentrations of 0.05% to 0.1 %, which are less than 0.2% claimed in the instant application (Abstract; Introduction; p. 4, sub-section 2.2, ¶ [0001] “DI water was used as the Newtonian solution, and an aqueous solution of PEO was selected as the viscoelastic solution. The PEO solution (Mw = ~2 MDa, Sigma-Aldrich, Saint Louis, MO, USA) was prepared by dissolving PEO in DI water to obtain a 0.05 wt% PEO (PEO500) solution. Then, fluorescent polystyrene (PS) particles (Thermo Scientific inc., Fremont, CA, USA) with particle sizes of 5 and 13 µm were added to these experimental solutions (0.05–0.1 wt% concentration), respectively.”). PEO is a known shear-thinning viscoelastic polymer in aqueous solution.).
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a method for detecting cell aggregates of biological cells using a suspension comprising viscoelastic fluid comprising shear-thinning polymer (e.g. 0.1%) that overlaps with the disclosed instant application to incorporate Kwon et al.’s teachings and select a higher molecular weight shear-thinning polymer within the known viscoelastic regime (e.g. ~2 MDa taught by Kwon et al.). Doing so would be a matter of routine optimization of known techniques to achieve a predictable viscoelastic flow. The inventions lie in the same fields of endeavor of quantitative analysis using viscoelastic microfluids. Motivation to combine includes to, “serve as a promising microfluidic pretreatment platform for flow cytometry.” (Kwon et al., p. 3, line 9-10).
Regarding Claim 39,
Hayden et al. and Dubois et al. teach the limitations of claim 25
Hayden et al. further teaches: wherein taking the one or more phase shift images of the biological cells in the suspension using the quantitative phase-contrast microscope comprises taking a first phase shift image of the biological cells in the suspension (Hayden et. al teaches a microfluidic system configured for quantitative phase imaging (e.g. digital holographic micrsoscopy) of biological cells flowing in suspension through a microfluidic chamber and capturing phase images of biological entities (e.g. cells) positioned within the imaging region (Abstract; ¶ [0036] & ¶ [0087]; ¶¶ [0023]- [0028])) at one or both of a first flow velocity of the suspension and a first shear rate in the suspension (Hayden et al. teaches pumping and pressure systems for microfluidic flow ¶ [0063])
Hayden et al. and Dubois et al. fail to disclose acquiring phase images at multiple different flow velocities and shear rates for comparison.
In a related art, Kwon et al. teaches: microfluidic flow of particle suspension in viscoelastic polymer solutions and discloses operating microfluidic system under controlled flow rate conditions while acquiring images. Specifically, Kwon et al. states, “p. 4, sub-section 2.2, ¶ [0002] “The flow rates were controlled with a syringe pump (LEGATO 111, KDScientific Inc., Holliston, MA, USA) and the images were captured 4 cm downstream from the inlet using CMOS camera... To allow the particle focusing behavior to be precisely analyzed, bright-field images and fluorescent images were capture…”). This disclosure demonstrates that flow rate is a controllable operating parameter of the microfluidic system, and images are acquired during the flow at selected flow rates. Kwon et al. further explains that particle migration behavior in viscoelastic flow depends on flow conditions, which inherently correspond to changes in shear rate within the microchannel. Accordingly, Kwon et al. teaches operation of a pump-driven microfluidic system at selectable flow velocities, and imaging of particles under those flow conditions.
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to operate Hayden et al. and Dubois et al.’s quantitative phase imaging system at multiple different flow velocities and corresponding shear rates, as taught by Kwon et al., because flow velocities and shear rates are known controllable variables in microfluidic systems. Shear rate in a microchannel is directly related to flow velocity, thus, adjusting the flow rate will produce a different shear rate. A person of ordinary skill in the art at the time of the invention would have recognized that a pump driven microfluidic imaging system, as taught by Hayden et al., is inherently capable of being operated at multiple selectable flow rates and that phase images may be acquired at each different rate. Varying flow velocity to obtain images under different shear and velocity conditions is routine operation of a microfluidic system and requires no structural modification to Hayden et al. beyond selection of a different pump setting, as taught by Kwon et al. The inventions lie in the same fields of endeavor of microfluidic quantitative analysis using viscoelastic microfluids. Combining Kwon’s teachings regarding controllable pump driven flow with Hayden’s phase imaging system merely applies known microfluidic operating parameters to a known imaging device to achieve predictable operation under different flow velocities and corresponding shear rates.
Claim 38 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), and in further view of Nam et al. (“Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid”, 2012; Copy was provided by applicant in the instant application and IDS).
Regarding Claim 38,
Hayden et al. and Dubois et al. teach the limitations of claim 25, including a microfluidic flow channel configured for quantitative phase-contrast imaging of cells, where cells are aligned within a “desired region” corresponding to the optical depth of the field of view of the imaging device and the biological cells are confined to a region and defined depth of view suitable for inspection (Hayden et al. ¶¶ [0023]- [0028]; Hayden et al. ¶¶ [0057]). Thus, Hayden et al. teaches positioning flowing cells within a focal region of a quantitative phase imaging device.
Hayden et al. and Dubois et al. fail to teach adapting viscoelastic or hydrodynamic focusing such that single cells are suspended in a single-cell stream to ensure focal plane alignment during channel flow.
In a related art, Nam et al. teaches: viscoelastic focusing in a microfluidic channel using a non-Newtonian fluid polymer solution to continuously focus particles into a narrow equilibrium stream (Abstract). Nam et al. teaches the elastic and inertial lift forces in the shear-thinning polymer solution drive particles toward equilibrium positions within the channel, producing a focused particle stream suitable for downstream analysis (p. 1349, sub-section “Principle of this study: elasto-inertial particle separation” ¶ ¶ [0001]- [0004]; p. 1353, sub-section “Conclusions”, ¶ [0001].)
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of viscoelastic flow positioning system using a quantitative phase-contrast microscope to incorporate the elasto-inertial focusing techniques of Nam et al. in order to achieve a more confined and predictable single-cell stream positioned within the focal plane of the quantitative phase-contrast microscope. Confining flowing cells to a focal plane is a known requirement in flow-based imaging methods, and the use of known viscoelastic focusing systems to achieve lateral confinement is a predictable application for improving microfluidic imaging. The combination merely applies a known focusing method taught by Lim et al., to a known imaging system taught by Hayden et. al. to obtain a predictable cell focused imaging. Hayden et al., Dubois et al., and Nam et al. fall in the same field of endeavor of quantitative biological cell analysis using microscopy. The motivation to combine would be to increase the scope of quantitative biological cell data rendered from quantitative phase-contrast microscopes.
Claim 40 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), and in further view of Rich (US 20130091937 A1).
Regarding Claim 40,
Hayden et al. teaches: a device for detecting cell (¶ [0002] “The present invention relates to techniques for aligning a non-spherical biological entity flowing in a sample that is to be inspected by an imaging device.”; Includes a system to achieve the task, see ¶ [0008]), the device comprising:
a mount configured to receive a microfluidic system comprising a measurement volume (Fig. 1; ¶ [0009] “The flow cell includes a flow chamber, a bottom flow input module, a top flow input module, a sample input module and an acoustic transducer.”; ¶ [0010] The bottom flow input module receives a first viscoelastic fluid and provides the first viscoelastic fluid to the flow chamber”; ¶ [0035] “Thus the aligning of the non-spherical biological entity in the depth of field of the interferometry microscopy device is achieved and this in turn leads to obtaining of high quality or focused images…volumetric measurements of components of the non-spherical biological entity…”; The flow cell is interpreted as equivalent to a microfluidic system because it comprises a measurement volume (e.g. flow chamber or microfluidic channel, volume of fluid measurements) and because it includes fluid connectors, it’s configured to position the microfluidic system relative to the microscope, and it includes the first flow chamber (a microfluidic chamber), as described in ¶ [0036] of the instant application. Additionally, the physical flow cell seen in Fig. 1 is interpreted as a mount because it is configured to receive the flow cell system.);
a microscope (¶ [0013] “The quantitative phase-contrast microscope may for example be a ptychographic imaging device or a digital holographic microscope…”) configured to take phase (¶ [0087] “By using the present technique and applying it to image RBCs 4 using digital holographic microscopy device 90, holograms, i.e., phase image … is recorded”; ¶ [0035] “Thus the aligning of the non-spherical biological entity in the depth of field of the interferometry microscopy device is achieved and this in turn leads to obtaining of high quality or focused images…, for example, volumetric measurements of components of the non-spherical biological entity…”; ¶ [0023] “…the flow chamber is a microfluidic channel…”; The flow chamber is equivalent to the measurement volume because it is a microfluidic channel, as described in ¶ [0036] of the instant application. Accordingly, the device is configured to take phase images of biological entities (e.g. cells, see ¶ [0054]) in the measurement volume.);
a microfluidics unit configured to receive a sample fluid comprising biological cells from a blood sample (¶ [0054] “…the sample with its components, i.e., one or more non-spherical biological entities such as red blood cells…”; ¶ [0011] “The sample input module receives the sample and provides the sample to the flow chamber…”; The sample unit (found in the flow system) is configured to perform the task of receiving a sample fluid comprising biological cells from a blood same, therefore is considered a microfluidics unit.), wherein the microfluidics unit is configured to generate a flow of the sample fluid through the measurement volume to one or both of viscoelastically and hydrodynamically focus cell (¶ [0011] “the sample laminarly flows in the flow chamber in the form of a sample laminar flow from one end of the flow chamber towards another end of the flow chamber.”; Abstract “The first and the second viscoelastic fluids laminarly flow along a bottom and a top wall of the flow chamber and the sample laminarly flows sandwiched between them. By controlling rate of flow of the first and/or the second viscoelastic fluids the sample flow, and thus the non-spherical biological entity, is focused in the desired region.”); and
(Interference patterns ¶ [0057] are found in phase images of red blood cells (RBC), taught in ¶ [0087]. Hayden et al. also teaches interference pattern includes RBC characteristics such as physical structures, morphology, etc. and the imaging device detects and analyzes the interference patterns (refer to ¶ [0083]). Examiner interprets identifying cells in the one or more phase images to be equivalent to detecting and analyzing red blood cells found in phase images of interference patterns. Hayden et al. further teaches the biological entities may include platelets, ¶ [0032] “The non-spherical biological entity may be, but not limited to, an erythrocyte, a platelet, an irregularly shaped leukocyte, and so on and so forth.” As previously established, the sample fluid consists of biological entities and is analyzed in in the flow chamber by the microscope using phase images (¶ [0054]; ¶ [0011]; Abstract; ¶ [0087]). Accordingly, the device is configured to identify platelets from phase images of the sample fluid flow obtained from the microscope, as recited in the claim.).
Hayden et al. fails to explicitly disclose detecting or identifying cell aggregates, including platelet aggregates, and while Hayden et al. teaches phase images, Hayden et al. fails to explicitly teach phase shift images. Hayden also fails to explicitly disclose using a controller.
In a related art, Dubois et al. teaches: a method for quantitative analysis of blood cells, platelets, or aggregates of cells and platelets using digital holographic microscopy (Abstract; ¶ [0010]) and quantitative phase contrast imaging (see ¶ [0025]). Dubois et al. further teaches detecting (see Dubois et al. ¶ [0034] “the recorded fields of view by the DHM show aggregates”; ¶ [0042] “the regions covered by the aggregates were detected.”), determining aggregate information from a corresponding image (¶ [0034] “…measure the aggregate shapes thanks to the quantitative phase contrast imaging capability of the DHM.”; ¶ [0046] “The volume of the aggregate…is obtained by computing in the corresponding phase image”). Dubois et al. also expressly teaches that “…using a flow chamber directly on the DHM” platelet aggregate formation may be dynamically studied (¶ [0053]). See also claim 8, which recites DHM analysis of blood platelet aggregates, and dependent claim 9, which separately limits the platelet aggregates to those adhering on a surface. The phase images used by Dubois et al. represent optical phase information obtained by quantitative phase contrast DHM and therefore correspond to the claimed phase shift images.
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to modify Hayden et al.’s focused, flow-based DHM device to identify platelet aggregates in the phase images using the platelet-aggregate detection and analysis taught by Dubois et al. Hayden et al. provides an arrangement for focusing biological entities, including platelets, within the imaging region of a flow chamber, while Dubois et al. teaches DHM identification and analysis of platelet aggregates and expressly contemplates performing such analysis using a flow chamber directly on the DHM (Dubois ¶ [0053]). A person of ordinary skill in the art therefore would have had reason to apply Dubois et al.’s platelet aggregate analysis to Hayden et al.’s in-flow imaging device to permit platelet aggregates carried in the sample flow to be positioned in the imaging region and identified from the resulting phase images.
Hayden et al. and Dubois et al. fail to disclose a controller.
In a related art, Rich teaches: a controller with accessible memory and a processor that transmits data (¶ [0021] “the controller… includes a storage device 48 with accessible memory.”; ¶ [0023] “…connected to a processor and may receive and transmit information about the acquired data… In addition, the controller 30 may receive information about other characteristics”) and performs other flow-related operations (¶ [0018]).
Therefore, would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a device that detects biological cells and/or platelets using phase shift images from a quantitative phase-contrast microscope (e.g. digital holographic microscopy device) to use the component of a controller, known to one of ordinary skill at the time of the effective filling date as taught by Rich, to perform the task of identifying platelet aggregates in a phase shift image of the sample fluid flow obtained from the microscope. Hayden et al., Dubois et al., and Rich all teach identifying and quantifying fluid samples using flow-based detection. The motivation to combine inventions is to provide the hardware to effectively identify platelet aggregates and transmit acquired data with increased speed.
Claim 41 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of Dimarzio & Warger (US 20080032325 A1), in further view of Michelson et al. ("Circulating Monocyte-Platelet Aggregates Are a More Sensitive Marker of In Vivo Platelet Activation Than Platelet Surface P-Selectin: Studies in Baboons, Human Coronary Intervention, and Human Acute Myocardial Infarction", 2001; Copy was provided by applicant in the instant application and IDS), and in further view of Rich (US 20130091937 A1).
Regarding Claim 41,
Hayden et al., Dubois et al., and Rich teach the limitations of claim 40.
Dubois et al., DiMarzio & Warger, and Michelson et al. teach: to determine one or more of a total number or fraction of platelet aggregates in the phase shift image (Limitation equally mirrors limitation found in Claim 26, lines 1-2. Refer to Claim 26 above for teachings and claims 25 and 26 for motivation to combine),
a number or fraction of platelet aggregates comprising at least a predefined number of cells in the phase shift image and (Limitation equally mirrors limitation found in Claim 29, lines 1-2. Refer to Claim 29 above for teachings and claims 25 and 29 for motivation to combine)
a total number or fraction of aggregated platelets that are contained in cell aggregates of any type in the phase shift image (Limitation equally mirrors limitation found in Claim 34, lines 2-3. Refer to Claim 34 above for teachings and claims 25 and 34 for motivation to combine).
It would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a device that detects biological cells and/or platelets using phase shift images from a quantitative phase-contrast microscope (e.g. digital holographic microscopy device) to determine quantitative platelet aggregate information taught by Dubois et al., accounting for mixed cell aggregates taught by Michelson et al., using predetermined numbers taught by DiMarzio & Warger. Hayden et al., Dubois et al., Rich, Michelson, and DiMarzio & Warger all teach identifying and quantifying fluid samples using flow-based detection. The motivation to combine inventions is to improve speed and accuracy of hardware and devices used for quantitative biological cell analysis.
Claim 42 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of DiMarzio & Warger (US 20080032325 A1), in further view of Mahan & Stewart (AU 2002233923 A1), in further view of Michelson et al. ("Circulating Monocyte-Platelet Aggregates Are a More Sensitive Marker of In Vivo Platelet Activation Than Platelet Surface P-Selectin: Studies in Baboons, Human Coronary Intervention, and Human Acute Myocardial Infarction", 2001; Copy was provided by applicant in the instant application and IDS) and in further view of Rich (US 20130091937 A1).
Regarding Claim 42,
Hayden et al., Dubois et al., and Rich teach the limitations of claim 40, including a controller.
Hayden et al., Dubois et al., DiMarzio & Warger, Mahan & Stewart, and Michelson et al. teach: to identify leukocyte-platelet aggregates in the phase shift image (Limitation equally mirrors limitation found in Claim 30, lines 2-3. Refer to Claim 30 above for teachings and claims 25 and 30 for motivation to combine),
to determine one or more of a total number or fraction of leukocyte-platelet aggregates in the phase shift image, a number or fraction of leukocyte-platelet aggregates comprising at least a predefined number of cells in the phase shift image (Limitation equally mirrors limitation found in Claim 31, lines 2-5. Refer to Claim 31 above for teachings and claims 25 and 31 for motivation to combine),
and a number or fraction of leukocyte-platelet aggregates comprising at least a predefined number of leukocytes in the phase shift image (Limitation equally mirrors limitation found in Claim 32, lines 1-3. Refer to Claim 32 above for teachings and claims 25 and 32 for motivation to combine).
It would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a device that detects biological cells and/or platelets using phase shift images from a quantitative phase-contrast microscope (e.g. digital holographic microscopy device) to determine quantitative platelet aggregate information taught by Dubois et al., accounting for the identification of leukocyte-platelet aggregates taught by Mahan & Stewart and determining quantitative information of leukocyte-platelet aggregates when a predefined condition (e.g. number of leukocytes) is implemented, as taught by Michelson et al.. One of ordinary skill at the time of the effective filing date could have incorporated a known controller taught by Rich to complete the tasks recited in claim 42. Hayden et al., Dubois et al., DiMarzio & Warger, Mahan & Stewart, Michelson et al., and Rich all teach identifying and quantifying fluid samples using flow-based detection and/or analysis. The motivation to combine inventions is to improve speed and accuracy of hardware and devices used for quantitative biological cell analysis.
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Hayden et al. (US 20190113433 A1) in view of Dubois et al. (US 20180114315 A1), in further view of Rich (US 20130091937 A1), and in further view of Holmes et al. (US 20140038206 A1).
Regarding Claim 43,
Hayden et al., Dubois et al., and Rich teach the limitations of claim 40, including a controller.
Hayden et al., Dubois et al., and Holmes et al. teach: to determine one or more of a size distribution of a plurality of cells, one or more parameters pertaining to said size distribution and a granularity measure from the phase shift image, wherein the granularity measure characterizes a granularity of one or more cells in the phase shift image ((Limitation equally mirrors limitation found in Claim 35, lines 2-4. Refer to Claim 35 above for teachings and claims 25 and 35 for motivation to combine).
Therefore, it would have been obvious to a person of ordinary skill in the art prior to the effective filling date of the claimed invention to have modified Hayden et al. and Dubois et al.’s teachings of a device for quantitative analysis of biological to incorporated Holmes et al.’s known teachings of determining sizes of cells and a size distribution or granularity of a plurality of cells and at least one parameter, including a mean of the Gaussian, a variance, a volume, a width, or an area of the base of cells, using phase shift images. One of ordinary skill at the time of the effective filing date could have incorporated a known controller taught by Rich to complete the tasks recited in claim 42. Doing so would increase the amount of data derived from a quantitative biological cell method and improve a user’s (e.g. physician) basis for analysis, diagnosis, etc. The inventions lie in the same field of endeavor of quantitative biological cell analysis using microscopes.
Conclusion
THIS ACTION IS MADE FINAL. 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 nonprovisional extension fee (37 CFR 1.17(a)) 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 SAMUEL DAVID BAYNES whose telephone number is (571)272-0607. The examiner can normally be reached Monday - Friday 8:00 am - 5:00 pm.
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, Stephen R Koziol can be reached at (408)918-7630. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/SDB/
Samuel D. Baynes
Examiner, Art Unit 2665
/Stephen R Koziol/Supervisory Patent Examiner, Art Unit 2665
Prosecution Insights