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 .
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.
Priority
This application is the U.S. National Stage (371) application of PCT/EP2019/055142 filed on 03/01/2019.
Claim Status
Claims 1, 11 and 14-16 are cancelled at the Applicant’s request. Claims 4-5 and 17 are currently amended, and the Applicant notes that no new matter is added. Claims 2-3, 6-10, 12-13 and 18 are previously presented. Claim 19 is new, and the Applicant notes that no new matter is added.
Thus, claims 2-10, 12-13 and 17-19 are pending and are under examination.
Withdrawn Objections & Rejections
The previous objections to claims 4-5 and 17, regarding informalities, are withdrawn in light of Applicant’s amendments of claims.
Maintained Rejections
Claim Rejections – 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 4 and 19 are newly included in this rejection.
Claims 4-5 and 19 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 4, the amended claim recites “the first calibration factor being a ratio between a measurement signal strength of the at least one first calibrator and a measurement signal strength of a first calibrator having a target structure”. It is not clear from the amendments if the first calibrator having the target structure is of the first or the second target structure type as previously recited in claim 17 onto which claim 4 depends. The specification only recites a “first reference calibrator having a target structure” but it does not provide what type of a target structure is present on the first calibrator (Page 7, lines 20-22; page 8, lines 15-17). Furthermore, the specification refers to the first reference calibrator as having the target structure and not the first calibrator. As such, the language of claim 4 is not clear, and thus claim 4 is deemed indefinite.
Regarding claim 5, it is not clear how the amendments match “kinds” with a “first type’ and “second type” of calibrator. It is even more confusing on why would the claim recite first kind versus first type and if there is any difference between reciting first type or first kind. Therefore, claim 5 is deemed indefinite.
Regarding claim 19, the claim recites the term “two types kinds”, and it is not clear what is meant by “types kinds”. It is even more confusing on why would the claim recite first kind versus first type and if there is any difference between reciting a first type or first kind. Therefore, claim 19 is deemed indefinite.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art (PHOSITA) to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 2-3, 6-10, 12-13 and 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Tei et al. (US 2010/0015643 A1) and Li et al. (US 2017/0315122 A1).
Claim 4 is new claims and is added to the current rejection of claims.
Regarding independent claim 17, the claim recites:
‘’ A flow cytometry measuring method comprising:
providing an analysis medium comprising a fluid and biological cells;
bringing at least one labeling molecule into contact with the analysis medium,
wherein the labeling molecule binds to at least one target structure located on a surface of any of
the cells that have the at least one target structure;
providing at least one first and at least one second calibrator, wherein
the at least one first and at least one second calibrator comprise solid particles of a
water-insoluble, inorganic and/or polymeric material,
a material, shape, and size of the at least one first calibrator is the same as a material,
shape, and size of the at least one second calibrator,
the at least one target structure is immobilized on a surface of the at least one first
calibrator, and
none of the at least one target structure is immobilized on a surface of the at least
one second calibrator;
mixing the at least one first and the at least one second calibrator with the analysis
medium, wherein the at least one labeling molecule located in the analysis medium binds to the at
least one target structure of the at least one first calibrator;
flowing the analysis medium through a measurement region of an energy beam
and/or an electric field, wherein the cells and the calibrator particles flow individually through the
measurement region;
capturing a first flow cytometry measured value for a first physical parameter of
the individual cells and the individual calibrator particles as the individual cells and individual
calibrator particles pass through the measurement region and a second flow cytometry measured
value for a second physical parameter of the individual cells and the individual calibrator particles
as the individual cells and the individual calibrator particles pass through the measurement region,
wherein the at least one first physical parameter is fluorescence radiation emitted by the at least
one labeling molecule when excited with the energy beam or the electric field;
distinguishing captured measurements associated with a cell from captured
measurements associated with a calibrator particle using the second flow cytometry measured
value for the second physical parameter; and
determining a normalized first flow cytometry measured value for individual cells
based on the at least one first flow cytometry measured value for the at least one first calibrator,
the at least one first flow cytometry measured value for the at least one second calibrator, and the
at least one first flow cytometry measured value for the individual cell, wherein the at least one
first flow cytometry measured value for the at least one first calibrator, the at least one first flow
cytometry measured value for the at least one second calibrator, and the at least one first flow
cytometry measured value for the individual cell, are in each case measured in said analysis
medium,
wherein at least one first flow cytometry measured value is captured for multiple
measuring channels, for the cells, the at least one first calibrator, and the at least one second
calibrator,
wherein a number of different labeling molecules are provided and brought into
contact with the analysis medium, the number of measuring channels corresponds to the number
of different labeling molecules, and at least two types of first calibrators are provided,
wherein at least a first type of first calibrator has at least one first target structure
and at least one second target structure on the surface thereof,
wherein at least a second type of first calibrator has the at least one second target
structure on the surface thereof,
wherein the at least one first target structure corresponds to at least one first target structure of the cells and the at least one second target structure corresponds to at least one second target structure of the cells”.
Regarding claim 17, Tei teaches a flow cytometry measuring method (Abstract).
Tei teaches providing an analysis medium comprising a fluid and biological cells (Page 1, [0001]).
Tei teaches bringing at least one labeling molecule into contact with the analysis medium (Page 3, [0035]).
Tei teaches that the labeling molecule binds to at least one target structure located on a surface of any of the cells that have the at least one target structure (Page 3, [0035]).
Tei teaches providing calibration beads that bear a known amount of an antigen protein (Page 3, [0030-0031]) at least one first and at least one second calibrator.
Tei teaches that at least one first and at least one second calibrator comprise solid particles of a water-insoluble, inorganic and/ or polymeric material such as latex (Page 3, [0032]).
Tei teaches that the material, shape, and size of the at least one first calibrator is the same as the material, shape, and size of the at least one second calibrator (Page 3, [0032]). While Tei, does not describe the shape of the particles, it is inferred from the description of Tei’s invention that the particles are beads that are spherical.
Tei teaches that at least one target structure is immobilized on a surface of the at least one first calibrator (Page 3, [0030-0032]).
Tei teaches mixing at least one first and at least one second calibrator with the analysis medium (Page 3,[0035]).
Tei teaches that at least one labeling molecule located in the analysis medium binds to at least one target structure of at least one first calibrator (Page 3, [0035]).
Tei teaches that at least one first physical parameter is fluorescence radiation emitted by the at least one labeling molecule when excited with the energy beam or the electric field ([0009]; [0037]).
Tei teaches distinguishing captured measurements associated with a cell from captured measurements associated with a calibrator particle using the second flow cytometry measured value for the second physical parameter such as using a second fluorescence (Page 28, claims 1 and 2).
Tei teaches determining a normalized first flow cytometry measured value for individual cells based on at least one first flow cytometry measured value for at least one first calibrator, at least one first flow cytometry measured value for at least one second calibrator, and at least one first flow cytometry measured value for the individual cell (Pages 3-4, [0037]).
Tei teaches that the at least one first flow cytometry measured value for the at least one first calibrator, the at least one first flow cytometry measured value for the at least one second calibrator, and the at least one first flow cytometry measured value for the individual cell, are in each case measured in said analysis medium (Page 4, [0038], “In a second method, two or more groups of beads bearing known and different amounts of an antigen protein are caused to coexist with test cells, and a fluorescence-labeled antibody for antigen protein is allowed to react therewith and then analyzed, whereby production of a calibration curve and acquisition of fluorescence intensity values for determining the amount of antibodies bound to antigen protein on test cells can be achieved in an assay system of a single flow cytometer.”).
Tei teaches that at least one first flow cytometry measured value is captured for multiple measuring channels, for the cells, the at least one first calibrator, and the at least one second calibrator (Page 3, [0035]).
Tei teaches that a number of labeling molecules are provided and brought into contact with the analysis medium (Page 3, [0035]).
Tei teaches that at least two types of first calibrators are provided (Page 3, [0030-0031]).
Regarding claim 17, Tei teaches that at least a first type of first calibrator has at least one first target structure and at least one second target structure on the surface thereof (Page 6, [0057]; page 7, [0061]).
Tei teaches that at least a second type of first calibrator has the at least one second target structure on the surface thereof (Page 3, [0031], “second group of beads bearing the antigen protein in an amount 10 times (i.e., x10) that of the first group”).
Tei teaches that the at least one first target structure corresponds to at least one first target structure of the cells (Page 6, [0057]).
Tei teaches that the at least one second target structure corresponds to at least one second target structure of the cells (Page 6, [0057]).
Regarding claim 2, Tei teaches determining the normalized first flow cytometry value by calculating a ratio (Page 3, [0035], “creating a calibration curve by use of the counts and the mixing ratios of the labeled antigen-protein-binding substance and the non-labeled anti-gen-protein-binding substance”).
Regarding claim 3, Tei teaches multiple first calibrators and multiple second calibrators are provided and mixed with the analysis medium (Page 3, [0030-0031]).
Tei teaches an averaged first flow cytometry measured value for the first calibrators is determined from the first flow cytometry measured values of the first calibrators (Page 3, [0030-0031]).
Tei teaches an averaged first flow cytometry measured value for the second calibrators is determined from the first flow cytometry measured values of the second calibrators (Page 3, [0030-0031]).
Tei teaches determination of the normalized first flow cytometry measured value for an individual cell comprises calculating a ratio (Pages 3-4, [0037], “the number of the antigen protein antibody-recognition sites per test cell (site/cell) can be numerically expressed and normalized.”).
Regarding claim 6, Tei teaches that the analysis medium contains at least two different populations of cells such as different types of leukocytes or monocytes with different expression of Toll-like receptors (Page 28, claim 6 and claim 7).
Tei teaches that there could be different population of cells to detect such as subpopulations of leukocytes that could have different signal strength (Page 4, [00369-0040]).
Tei teaches that the signal strength of the first flow cytometry measured values of the first calibrator type lies between the first signal strength range and the second signal strength range from the calibration curve (Page 3, [0030] and [0035]). A skilled artisan would have known that when a calibration curve is being made, it should span a range to cover the first or lowest signal strength and the highest or lowest signal strength. Moreover, Tei notes that a calibration curve is prepared to cover the possible fluorescent intensities while measuring an analyte with flow cytometry (Page 1, [0008]).
Regarding claim 7, Tei A teaches that the largest dimension of the solid particles of at least one first calibrator and at least one second calibrator is less than 20 µm and/or the smallest dimension of the solid particles of at least one first calibrator and at least one second calibrator is greater than 4 µm (Page 11, [0085]).
Regarding claim 8, Tei teaches that the material of the solid particles is latex (Page 3, [0032]).
Regarding claim 10, Tei teaches that at least one first flow cytometry measured value is captured for multiple measuring channels for the cells, at least one first calibrator and at least one second calibrator (Page 3, [0035]).
Tei teaches that labeling molecules are provided and brought into contact with the analysis medium (Page 3, [0035]).
Regarding claim 10, Tei teaches that at least one first calibrator has a number of types of calibrators corresponding to the number of measurement channels (Page 3, [0030-0031]).
Regarding claim 12, Tei teaches that the target structure on the at least one first calibrator has at least one antigen (Page 3, [0030-0032]).
Regarding claim 13, Tei teaches that the target structure of the at least one first calibrator is immobilized on the solid particle of the first calibrator via at least one activated carboxy group and/or at least one activated NH2 group (Page 3, [0034]).
Regarding claim 18, Tei teaches that the antigen comprises at least one protein or receptor (Page 3, [0030-0032]).
Regarding claim 17, Tei does not teach that the labeling molecules brought in contact with the analysis medium are of different types.
Tei does not teach that the number of measuring channels corresponds to the number of different labeling molecules.
Tei does not teach that each type of calibrator specifically binding to one of the different labeling molecules.
Tei does not teach flowing the analysis medium through a measurement region of an energy beam and/or an electric field.
Tei does not teach that the cells and the calibrator particles flow individually through the measurement region.
Tei does not teach capturing a first flow cytometry measured value for a first physical parameter of the individual cells and the individual calibrator particles as the individual cells and individual calibrator particles pass through the measurement region.
Tei does not teach capturing a second flow cytometry measured value for a second physical parameter of the individual cells and the individual calibrator particles as the individual cells and the individual calibrator particles pass through the measurement region.
Regarding claim 9, Tei does not teach that at least one second flow cytometry measured value is a forward scatter measured value for a forward scatter of the energy beam occurring within the measurement region and/or a sideward scatter measured value for a sideward scatter of the energy beam occurring in the measurement region.
Regarding claim 10, Tei does not teach providing different labeling molecules and bringing them in contact with the analysis medium.
Regarding claim 10, Tei does not teach that the number of measuring channels corresponds to the number of different labeling molecules.
Regarding claim 10, Tei does not teach that each type of calibrator specifically binding to one of the different labeling molecules.
Regarding claim 17, Li teaches flowing the analysis medium through a measurement region of an energy beam and/or an electric field (Page 1, [0003]; page 3, [0016]).
Li teaches that the cells and the calibrator particles flow individually through the measurement region (Page 4, [0024]).
Li teaches capturing a first flow cytometry measured value for a first physical parameter of the individual cells and the individual calibrator particles as the individual cells and individual calibrator particles pass through the measurement region (Page 4, [0024] and [0028]). Li states that a flow channel is mounted in the housing and coupled to a flow cytometer for hydrodynamic focusing of calibration beads or cells to generate a flow cytometry value.
Li teaches capturing a second flow cytometry measured value for a second physical parameter of the individual cells and the individual calibrator particles as the individual cells and the individual calibrator particles pass through the measurement region (Page 4, [0024] and [0028]). Li states that a flow channel is mounted in the housing and coupled to a flow cytometer for hydrodynamic focusing of calibration beads or cells to generate a flow cytometry value.
Li teaches using a plurality of fluorescent labels that are different as labeling molecules (Page 4, [0028]; page 11, [0091]).
Li teaches that the number of measuring channels corresponds to the number of different labeling molecules (Page 13, [0099]).
Li teaches that each type of calibrator specifically binding to one of the different labeling molecules (Page 6, [0048-0049]).
Regarding claim 9, Li teaches that at least one second flow cytometry measured value is a forward scatter measured value for a forward scatter of the energy beam occurring within the measurement region (Page 10, [0082]).
Regarding claim 10, Li teaches that a number of different labeling molecules are provided and brought into contact with the analysis medium (Page 11, [0091]).
Li teaches that the number of measuring channels corresponds to the number of different labeling molecules (Page 13, [0099]).
Li teaches that each type of calibrator specifically binding to one of the different labeling molecules (Page 6, [0048-0049]).
It would have been obvious for a PHOSITA before the effective filing date of the application to combine the calibration factor of Li with the flow cytometric method of Tei to detect a target cell or protein because Li introduced the ability to use a calibration factor to further automate the method of flow cytometry measurements (Page 3, [0018]) and noted that a predetermined calibration factor would allow for the improvement of linear dynamic range of measurement (Page 3, [0018]). Furthermore, Tei offered a calibration kit for the flow cytometry measurement from which a calibration curve is made (Page 4, [0041-0045]). Thus, a skilled artisan would have been motivated to combine the above methods and inventions for convenience. A PHOSITA would have had a reasonable expectation of success in combining the methods of Li and Tei because the methods are directed to detecting a target by flow cytometry.
It would have been obvious for a PHOSITA to combine the calibration factor of Li with the kit of Tei to further automate the detection of a target by a flow cytometric method.
Regarding claim 17, A skilled artisan would have understood from Tei, that different antigenic determinants could be employed on the surface of the beads for making the calibration curve to account for the presence of different recognition sites for the antibody on cells (Page 6, [0057],“When standard beads whose particle size or TLR2 binding manner is changed are employed or when an anti-TLR2 antibody having a different recognition site is employed”).
Regarding claims 2-3, Tei does not teach calculating a ratio of a difference between the averaged first flow cytometry measured value for the first calibrators and the averaged first flow cytometry measured value for the second calibrators to a difference between the first flow cytometry measured value for the individual cell and the averaged first flow cytometry measured value for the second calibrators.
However, a skilled artisan would have made such calculations based on the type of calibrators used in making the calibration curve.
Response to Arguments
Applicant's arguments filed 03/09/2026 have been fully considered but they are not persuasive in regards to the rejection of claims 2-3, 6-10, 12-13 and 17-18 under 35 U.S.C. 103, regarding obviousness as being unpatentable over Tei et al. (US 2010/0015643 A1) and Li et al. (US 2017/0315122 A1).
The Applicant argued that Applicant's remarks regarding Tei et al. and Li et al. as made in the preceding Amendment dated 25-August-2025 (which was in response to the final Office Action dated
23-April-2025) are incorporated herein by reference in their entirety.
Applicant’s arguments were fully considered but are unpersuasive for the reasons set forth in the previous Office action dated 09/10/2025 on pages 15-16.
The Applicant argued that Tei et al. and Li et al., alone or in any combination, provide no
disclosure or suggestion with regard to the measurement of cells and calibrators in the same
analysis medium (under identical conditions) for purposes of determining or otherwise generating
normalized values, such as a normalized first flow cytometry measured value for individual cells.
This argument is not persuasive because a skilled artisan would have known to account for the matrix effect to generate a valid calibration curve. It is well known in the art of flow cytometry that the same medium and conditions should be used in measuring cells and calibrators to generate mean fluorescence intensities (MFI) that are comparable as noted by Mittag et al. (J. Biophoton. 2, No. 8–9, 470–481 (2009), page 475, left column, third paragraph, “For standardization of fluorescence intensities, MFI values are converted into ABC (antibody binding capacity). For that purpose, beads with a defined number of antibody binding sites on the surface (Table 1) are used. These beads are treated in the same way as cells and stained with the same antibodies”). And as such, Mittag also teaches that beads that are used for calibration are measured with the same settings that are used for analyzing biological materials (Page 472, left column, last paragraph, “This calibration can be done by beads”; page 472, right column, second paragraph, “Prior to analysis, beads are diluted as specified by the manufacturer and approximately 10 000 events should be measured with exactly the same settings as used for the analyzed biological material.”). Furthermore, Tei teaches that the at least one first flow cytometry measured value for the at least one first calibrator, the at least one first flow cytometry measured value for the at least one second calibrator, and the at least one first flow cytometry measured value for the individual cell, are in each case measured in the same analysis medium (Page 4, [0038], “In a second method, two or more groups of beads bearing known and different amounts of an antigen protein are caused to coexist with test cells, and a fluorescence-labeled antibody for antigen protein is allowed to react there with and then analyzed, whereby production of a calibration curve and acquisition of fluorescence intensity values for determining the amount of antibodies bound to antigen protein on test cells can be achieved in an assay system of a single flow cytometer”).
Therefore, the previous rejection of claims 2-3, 6-10, 12-13 and 17-18 under 35 U.S.C. 103, regarding obviousness, is maintained and is made final.
The Applicant’s amendments to claim 4 did not overcome the previous rejection of claim 4 under 35 U.S.C. 112(a) because the amendments necessitated a new ground of rejection as previously discussed.
The Applicant’s amendments to claim 5 did not overcome the previous rejection of claim 5 under 35 U.S.C. 112(b) because the amendments necessitated the new ground of rejection as previously discussed.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any 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.
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.
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/OMAR RAMADAN/Examiner, Art Unit 1678
/GREGORY S EMCH/Supervisory Patent Examiner, Art Unit 1678
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