Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
Claims 1-3, 6-17, 19-20, 22-26, 30-35, 39-42, 44 and 73-76 are pending and under examination.
Priority
This application claims priority to 63/389,487 filed on July 15, 2022. The effective filing date of the current application is July 15, 2022.
Information Disclosure Statement
The information disclosure statements filed on August 3, 2023 and November 17, 2023 comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. All references were considered.
Drawings
The drawings are objected to because the figures are not properly labeled.
37 CFR 1.84 (u)(1) states “The different views must be numbered in consecutive Arabic numerals, starting with 1, independent of the numbering of the sheets and, if possible, in the order in which they appear on the drawing sheet(s). Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter. View numbers must be preceded by the abbreviation "FIG." Where only a single view is used in an application to illustrate the claimed invention, it must not be numbered and the abbreviation "FIG." must not appear.”
In the instant case, the view numbers for Figures 1-9 are preceded by the word "Figure" instead of the abbreviation "FIG.".
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The abstract of the disclosure is objected to because it is very short and non-descriptive of the invention. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text.
Applicant is reminded of the proper content of an abstract of the disclosure.
A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art.
If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Where applicable, the abstract should include the following: (1) if a machine or apparatus, its organization and operation; (2) if an article, its method of making; (3) if a chemical compound, its identity and use; (4) if a mixture, its ingredients; (5) if a process, the steps.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
Claim Objections
Claim 12, 22 and 24 are objected to because of the following informalities:
Claim 12 recites “wherein the correlating the activity of G6DPH … comprising comparing the activity of the G6DPH to a standard curve” which is ungrammatical. It is suggested the limitation be amended to recite “wherein correlating the activity of G6DPH … comprises comparing the activity of the G6DPH to a standard curve”.
Claim 22 recites “activity of gluthione reductase” in lines 3 and 4, which is misspelled and should be amended to recite “activity of glutathione reductase”.
Claim 24 is missing a semicolon and conjunction at the end of line 6. The limitation should be amended to recite “lysed blood sample; and”.
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-3, 6-17, 19-20, 22-26, 30-35, 39-42, 44 and 73-76 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The judicial exception is a method (Step 1: Yes) of determining hematocrit in a blood sample using a mathematical correlation (Step 2A prong one: Yes) to measure the amount of NADPH, G6DPH, or reduced tetrazolium dye in a lysed blood sample and correlate the measured value to the hematocrit of the blood sample. The additional elements do not integrate the judicial exception into a practical application because they are mere data gathering and applying or using the judicial exception in a particular technological environment (Step 2A prong two: No).
The measurement of an analyte in a blood sample is well-understood, routine and conventional in the art (Step 2B: No), as shown by Veskoukis et al. (“Spectrophotometric assays for measuring redox biomarkers in blood and tissues: the NADPH network”, Redox Report, 2018, Vol. 23, No. 1, pp.47-56), Bossuyt et al. (“Comparative Analysis of Whole Blood Lysis Methods for Flow Cytometry”, Cytometry, 1997, Vol. 30, Issue 3, pp.124-133), and Anderson (US 10,254,292 B2 issued on April 9, 2019).
Veskoukis et al. teaches the detection of NADPH-related molecules including NADPH and NADH in blood and tissues (abstract). Veskoukis teaches the extraction of NADPH from tissue lysate, and detection of the remaining amount of NADP+ to NADPH (p.49, 2nd column last paragraph). Veskoukis further teaches that G6D and G6PD can be used to detect the conversion of MTT to Formazan and determine the concentration of NADPH (p.50, Figure 2).
Bossuyt et al. teaches a parallel evaluation of six whole blood lysis methods comparing light scatter and quantitative fluorescence intensity based on quantitative flow cytometry (abstract).
Anderson teaches estimating the relative amounts of identifiable compartments within a biological sample, for example providing a measurement of hematocrit from a dried blood sample (abstract). Anderson teaches measuring in a blood sample the number of molecules of a compartment-specific molecule in a proteolytic digest of said sample by quantitative mass spectrometry or at least one monitor peptide (Col. 76, Claim 20).
The steps of lysing the blood sample, measuring the amount of NADPH, G6PDH or reduced tetrazolium dye and correlating the measurement to hematocrit are mere instructions to apply an exception. This judicial exception is not integrated into a practical application (Step 2A prong two: No) because claims do not include any additional elements that are sufficient to amount to significantly more than the judicial exception (Step 2B: No). The additional steps of lysing blood and measuring an amount of NADPH, G6PDH or tetrazolium dye are routine and conventional activities well-understood in the art before the filing of the instant invention.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-3, 6-8, 10-14, 19, 22-26, 30, 33-35, 39 and 40 are rejected under 35 U.S.C. 103 as being unpatentable over Abbott et al. (US 4,699,887 published on October 13, 1987) in view of Euro et al. (WO 2022/008802 A1, published January 13, 2022).
Regarding claim 1, Abbott teaches a method for measuring hematocrit (abstract). Abbott teaches the hematocrit of a blood sample can be estimated by electrochemically measuring the concentration of an electrochemically detectable marker before and after the ratio of the red blood cell volume to the plasma volume has been decreased (col.2, lines 18-22). Abbott teaches that the ratio of red cell volume to the plasma volume can be decreased by lysing the red blood cells using detergent or sonication (col.2, lines 36-38). Abbott teaches measuring sodium ion concentration before and after lysing the red blood cells (col. 2, lines 41-44). Abbott teaches an equation to estimate the hematocrit using the sodium concentration before and after lysis (col.2, lines 53-62). Abbott teaches that if a marker other than Na+ is used, the term “Na+” in the equation will be replaced with “Marker”, and a more exact equation that takes into account the water content of the red cell volume and the plasma volume can also be used to calculate hematocrit (col.3, lines 7-25).
Abbott does not teach measuring NADPH, and correlating NADPH amount to hematocrit in the lysed blood sample.
However, Euro teaches fresh or frozen samples are added into pre-heated ethanol resulting in cell lysis and release of metabolites into the ethanol solution (p.43, lines 7-10). Euro teaches the supernatant from the centrifugation step comprises metabolites including NADPH (p.43, lines 11-13). Euro teaches that measurement of the metabolites can be performed on the same day (p.43, lines 13-14). Euro teaches analysis of NAD metabolites in whole blood (p.46, line 20). Euro teaches that development of simple, effective and specific method and tools for determining concentrations of all four NAD metabolites separately would provide a significant advance in the field of personalized medicine as it would allow targeted treatment of individuals who would benefit from treatments (p.3, lines 7-12). Detection mode of the method or kit of the present invention can be any conventional detection mode including electrochemical detection mode. In one embodiment, NAD metabolites can be detected or analyzed by liquid chromatography and/or mass spectrometry (e.g. LC-MS and/or HPLC) (p.27, line 35 to p. 28, line 4).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace sodium ions taught by Abbott with NADPH taught by Euro as the analyte molecule in the method of Abbott, because Euro teaches that NADPH can be measured in whole blood, and Abbott teaches other analytes can be used to correlate hematocrit (col.2, lines 18-22). One of ordinary skill in the art would have motivated to do so because Euro teaches that such a method is a simple effective method to determine the concentration of NAD metabolites. One of ordinary skill would have found it beneficial to do so because Euro teaches that being able to detect NAD metabolites individually would provide a significant advance in the field of personalized medicine.
Regarding claims 2 and 3, Abbott teaches potentiometric measurement with ion-selective electrodes or amperometric measurement (col.2, lines 23-25).
Abbott does not teach measuring UV-Vis absorption (claim 2) or measuring in the range of about 500 nm to about 600 nm (claim 3).
Euro teaches the amount of NADPH is determined after an enzymatic reaction by measuring change in absorbance of chromogen at an appropriate wavelength relating to the reagents used, such as wavelength 500-600nm (p.28, lines 12-15).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the potentiometric measurement taught by Abbott with UV-Vis absorption in the range of 500 nm to 600 nm, as taught by Euro. Each of Abbott and Euro teach the detection of an analyte (i.e. detectable marker) in blood samples. One of ordinary skill in the art would reasonably expect that replacing one known measurement tool with another would predictably result in the measurement of NADPH in a sample, and it was known in the art at the time of invention that NADPH could be measured using UV-Vis absorption in the range of 500nm – 600nm.
Regarding claim 6, Abbott teaches measuring the amount of sodium ion concentration in whole blood and lysed blood samples using an electrochemical electrolyte analyzer, calculating the hematocrit using equation 1, and comparing to the amount of hematocrit measured in a sample using a microhematocrit method (col.3, Example 1).
Abbott does not teach comparison of samples to a standard curve.
Euro teaches that after measuring the absorbances, the concentrations of NAD metabolites in the extract can be determined by comparison of the assay response of the standards whose concentration is known (p.28, lines 19-22).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to prepare a standard curve taught by Euro in the method of Abbott to accurately determine the concentration of the analyte in the sample. One of ordinary skill in the art would reasonably expect that preparing a standard curve of the analyte to be measured would predictably result in a method of determining the concentration of the analyte in an unknown sample, because it was known in the art at the time of invention that standard curves could be prepared and used for such purpose.
Regarding claim 7, Abbott teaches a method for measuring hematocrit (abstract). Abbott teaches the hematocrit of a blood sample can be estimated by electrochemically measuring the concentration of an electrochemically detectable marker before and after the ratio of the red blood cell volume to the plasma volume has been decreased (col.2, lines 18-22). Abbott teaches that the ratio of red cell volume to the plasma volume can be decreased by lysing the red blood cells using detergent or sonication (col.2, lines 36-38). Abbott teaches measuring sodium ion concentration before and after lysing the red blood cells (col. 2, lines 41-44). Abbott teaches an equation to estimate the hematocrit using the sodium concentration before and after lysis (col.2, lines 53-62). Abbott teaches that if a marker other than Na+ is used, the term “Na+” in the equation will be replaced with “Marker”, and a more exact equation that takes into account the water content of the red cell volume and the plasma volume can also be used to calculate hematocrit (col.3, lines 7-25).
Anderson does not teach measuring G6PDH or glutathione reductase.
However, Euro teaches fresh or frozen samples are added into pre-heated ethanol resulting in cell lysis and release of metabolites into the ethanol solution (p.43, lines 7-10). Euro teaches that measurement of the metabolites can be performed on the same day (p.43, lines 13-14). Euro teaches analysis of NAD metabolites in whole blood (p.46, line 20). Euro teaches enzymes suitable for the cyclic enzymatic reaction of the present invention (p.21, lines 11-12). Euro further teaches an NADP-specific enzyme is a glucose-6-phosphate dehydrogenase (G6PDH) (p.21, lines 21-22). Euro teaches an NADP+/NADPH colorimetric assay using glucose-6-phosphate dehydrogenase (p.48, V. NADP+/NADPH colorimetric assay). Euro teaches that development of simple, effective and specific method and tools for determining concentrations of all four NAD metabolites separately would provide a significant advance in the field of personalized medicine as it would allow targeted treatment of individuals who would benefit from treatments (p.3, 7-12).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace measuring sodium ion concentration taught by Abbott with measuring G6PDH or glutathione activity taught by Euro as the analyte in the method of Abbott, because Euro teaches that G6PDH or glutathione activity can be measured using an NADPH colorimetric assay of whole blood samples. One of ordinary skill in the art would have motivated to do so because Euro teaches that such a method is a simple effective method to determine the concentration of NAD metabolites. One of ordinary skill would have found it beneficial to do so because Euro teaches that being able to detect NAD metabolites individually would provide a significant advance in the field of personalized medicine.
Regarding claim 8, Abbott teaches potentiometric measurement with ion-selective electrodes or amperometric measurement (col.2, lines 23-25).
Abbott does not teach measuring a UV-Vis absorption.
Euro teaches that absorbance was measured at 573 nm using a plate reader.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the potentiometric measurement taught by Abbott with a UV-Vis measurement using a plate reader taught by Euro. Each of Abbott and Euro teach detecting an analyte in a blood sample. One of ordinary skill would reasonably expect that replacing one known detection method with another would predictably result in detecting G6PDH or glutathione activity in a blood sample.
Regarding claim 10, Abbott does not teach adding NADP+ to the sample prior to measuring the activity of G6PDH.
However, Euro teaches the supernatant from the centrifugation step comprises metabolites including NADPH (p.43, lines 11-13). Euro teaches a reagent comprising added NADPH and a GSH-specific enzyme such as glutathione reductase for determining amounts of GSH and/or GSSG [reduced or oxidized forms of glutathione] (p.25, lines 22-25). Euro further teaches measurements of concentrations of reduced and/or oxidized glutathione (GSH, GSSG), which ratio GSH/GSSG is directly dependent of NADPH/NADP+ balance (p.24, lines 5-7).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Abbott to further include adding NAD+ or NADP+ to the sample as taught by Euro, because Euro teaches that the measurements of reduced GSH/GSSG is directly depending on NADPH/NADP+ balance. One of ordinary skill in the art would have been motivated to add NADP+ to the reaction because the NADPH/NADP+ balance affects the measurement of GSH and GSSG resulting from glutathione reductase activity.
Regarding claim 11, Abbott teaches lysing blood samples using detergent or sonication (col.2, lines 36-38).
Regarding claim 12, Abbott teaches measuring the amount of sodium ion concentration in whole blood and lysed blood samples using an electrochemical electrolyte analyzer, calculating the hematocrit using equation 1, and comparing to the amount of hematocrit measured in a sample using a microhematocrit method (col.3, Example 1).
Abbott is silent on the comparison of samples to a standard curve.
Euro teaches that after measuring the absorbances, the concentrations of NAD metabolites in the extract can be determined by comparison of the assay response of the standards whose concentration is known (p.28, lines 19-22).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to prepare a standard curve taught by Euro in the method of Abbott to accurately determine the concentration of the analyte in the sample. One of ordinary skill in the art would reasonably expect that preparing a standard curve of the analyte to be measured would predictably result in a method of determining the concentration of the analyte in an unknown sample, because it was known in the art at the time of invention that standard curves could be prepared and used for such purpose.
Regarding claims 13 and 14, Abbott does not teach measuring the amount of G6PDH redox products in the blood sample (claim 13) or wherein the G6PDH redox product is NADPH (claim 14).
However, Euro teaches measurement of concentrations of reduced and/or oxidized glutathione (GSH, GSSG) (p.24, line 5-6). Euro teaches that the ratio of GSH/GSSG is directly dependent on NADPH/NADP+ balance, and measuring both ratios from the same sample allows accurate estimation of oxidative damage or stress in the body (p.24, lines 6-7 and 11-14). Euro identifies that G6PDH is involved in a reaction converting NADP to NADPH (Figure 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace measuring sodium ion concentration taught by Abbott with measuring NADPH taught by Euro as the analyte in the method of Abbott, because Euro teaches that NADPH results from the action of G6PDH. One of ordinary skill in the art would reasonably expect that measuring NADPH production in the sample would predictably result in measuring the amount of G6PDH redox products, because Euro teaches NADPH results from G6PDH activity.
Regarding claim 19, Abbott does not teach the rate of formation of NADPH is inversely correlated to the hematocrit in the blood sample.
Euro is silent on whether the rate of NADPH formation is inversely correlated to the hematocrit in the blood sample.
However, the relationship between NADPH formation and hematocrit of the blood sample is a mathematical relationship determined from the measurement of the NADPH in the blood sample. As evidenced by Scott et al., there is a strong inverse correlation between NADPH levels and methemoglobin concentration (p.2061, 2nd column 2nd paragraph).
Thus, one of ordinary skill in the art would reasonably expect that obtaining an NADPH measurement according to the method of Anderson and Euro would predictably result in an inverse correlation of NADPH formation to the hematocrit in the blood sample, because it was known in the art at the time of invention that NADPH and methemoglobin were inversely correlated.
Regarding claims 22 and 23, Abbott does not teach GSSG.
However, Euro teaches GSSG can be measured from the same biological samples to complement NADPH/NADP+ measurements (p.24, lines 15-16). Euro teaches contacting GSSG with a GSH-specific enzyme such as glutathione reductase in the presence of added NADPH and a chromogen for determining amount of GSSG (p.24, lines 32-35). Euro further teaches measuring the amount of GSSG and comparing the detected amount to a standard (p.42, lines 23-24).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace measuring sodium ion concentration taught by Abbott with GSSG taught by Euro as the analyte in the method of Abbott to determine glutathione reductase activity, because Euro teaches that glutathione reductase can be used for determining amounts of GSSG. One of ordinary skill in the art would reasonably expect that selecting GSSG as a known substrate for glutathione reductase would predictably result in determining the activity of glutathione reductase in a blood sample, because it was known in the art at the time of invention that GSSG amounts could be determined using glutathione reductase. One of ordinary skill in the art would reasonably expect that measuring the amount of GSSG in the blood sample and comparing the amount of GSSG with a standard curve would predictably result in determining the hematocrit of a blood sample, because Abbott teaches that different analytes can be measured and correlated to hematocrit in blood samples.
Regarding claim 24, Abbott teaches a method for measuring hematocrit (abstract). Abbott teaches the hematocrit of a blood sample can be estimated by electrochemically measuring the concentration of an electrochemically detectable marker before and after the ratio of the red blood cell volume to the plasma volume has been decreased (col.2, lines 18-22). Abbott teaches that the ratio of red cell volume to the plasma volume can be decreased by lysing the red blood cells using detergent or sonication (col.2, lines 36-38). Abbott teaches measuring sodium ion concentration before and after lysing the red blood cells (col. 2, lines 41-44). Abbott teaches an equation to estimate the hematocrit using the sodium concentration before and after lysis (col.2, lines 53-62). Abbott teaches that if a marker other than Na+ is used, the term “Na+” in the equation will be replaced with “Marker”, and a more exact equation that takes into account the water content of the red cell volume and the plasma volume can also be used to calculate hematocrit (col.3, lines 7-25).
Abbott does not teach adding a tetrazolium dye or measuring an amount of a reduction product of the tetrazolium dye in the lysed blood sample.
Euro teaches chromogen 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for determining amounts of NAD+, NADP+, NADH and NADPH (p.21, lines 28-29). Euro teaches an enzyme which specifically recognizes and uses NAD+ or NAD+ as substrate is used for the reaction producing NADH or NADPH (p.20, lines 21-23). Euro teaches each enzyme reduces an oxidized NAD metabolite and the reduced form then donates an electron to an electron carrier which then passes the electron to the final electron acceptor (e.g. tetrazolium bromide (MTT)); resulting in NAD(P)H converted back to the oxidized form and MTT reducing to formazan (p.20, lines 23-29). Euro teaches measuring absorption change of MTT upon its reduction (p.20, lines 30-31). Euro teaches their method allows extraction and analysis from very small amounts of sample, such as from a single drop of blood (p.3, lines 33-34.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the method of measuring sodium ion concentration of Abbott with a method of adding MTT to the blood sample and measuring the reaction product of the MTT taught by Euro, because Euro teaches using MTT to determine the amounts of NAD+, NADP+, NADH and NADPH. Each of Abbott and Euro teach detecting analytes in whole blood samples. One of ordinary skill in the art would reasonably expect that replacing one known assay method with another would predictably result in the correlation of NADPH in the blood sample with hematocrit, because Euro teaches the MTT assay can be used to determine NAD metabolites in blood samples. One of ordinary skill in the art would have found it beneficial to use a method that required very small amounts of sample, such as a single drop of blood to detect NAD metabolites from a blood sample.
Regarding claims 25 and 26, Abbott does not teach measuring the amount of reduction product of the tetrazolium dye in the lysed blood sample comprises measuring a UV-Visible absorption (claim 25) or measuring in the range between about 570 nm and 650 nm (claim 26).
However, Euro teaches the detection comprises measuring absorption change of MTT upon its reduction in the assay reaction (e.g., formazan can be detected at a wavelength 573 nm) (p.20, lines 29-32).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to measure the reduction product of MTT using UV-visible absorption at 573nm wavelength because Euro teaches that formazan crystals, the reduction product of MTT can be detected at 573nm wavelength. One of ordinary skill in the art would reasonably expect that using the known MTT assay method of measuring formazan crystal formation at 573nm would predictably result in measuring the amount of reduction product of the MTT dye, because the MTT assay and formazan measurement using absorption was well-known in the art at the time of invention.
Regarding claim 30, Abbott does not teach comparing the amount of the reduction product of the tetrazolium dye to a standard curve.
Euro teaches each standard was pipetted in duplicates on the plate starting from 0 point to produce standard curve in the assay (p.49, lines 4-5). Euro teaches that the method includes use of a standard curve based on known concentrations of commercially available standards used in the same assay together with analyzed samples, and determination of concentration of analyzed NAD metabolites is done by comparison of the assay response of a sample to that of the standards whose concentrations are known (p.29, lines 15-20).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to prepare a standard curve taught by Euro for the tetrazolium dye reaction product, because Euro teaches using a comparison of assay response to known concentration standards. One of ordinary skill would have been motivated to compare an amount of reduction product of the tetrazolium dye to a standard curve to determine what concentration of reduction product was present in each experimental sample.
Regarding claim 33, Abbott teaches lysing blood samples using detergent or sonication (col.2, lines 36-38).
Regarding claims 34 and 35, Abbott does not teach measuring a UV-Visible absorption (claim 34) or measuring in the range between about 570 nm and 650 nm (claim 35).
However, Euro teaches the detection comprises measuring absorption change of MTT upon its reduction in the assay reaction (e.g., formazan can be detected at a wavelength 573 nm) (p.20, lines 29-32).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have measured the reduction product of the MTT using UV-visible absorption at 573nm wavelength because Euro teaches that formazan crystals, the reduction product of MTT can be detected at 573nm wavelength. One of ordinary skill in the art would reasonably expect that using the known MTT assay method of measuring formazan crystal formation at 573nm would predictably result in measuring the amount of reduction product of the MTT dye, because the MTT assay and formazan measurement using absorption was well-known in the art at the time of invention.
Regarding claim 39, Abbott does not teach the tetrazolium dye is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.
Euro teaches chromogen 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for determining amounts of NAD+, NADP+, NADH and NADPH (p.21, lines 28-29).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to select 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) as the tetrazolium dye because Euro teaches a colorimetric assay to detect NADPH using MTT. One of ordinary skill in the art would reasonably expect that selecting a common tetrazolium dye such as MTT would predictably result in a detectable colorimetric change that could be quantified using standard assay techniques, because the MTT colorimetric assay for NADPH measurement was known in the art at the time of invention.
Regarding claim 40, Abbott teaches freshly collected whole blood samples (col.3, Example 1).
Claims 9, 41-42, 44 and 73-76 are rejected under 35 U.S.C. 103 as being unpatentable over Abbott et al. (US 4,699,887 published on October 13, 1987) in view of Euro et al. (WO 2022/008802 A1, published January 13, 2022) as applied to claim 1 above, and further in view of Anderson (US Patent No. 10,254,292, B2 issued on April 9, 2019).
The teachings of Abbott et al. and Euro et al. are discussed above.
Regarding claim 9, Abbott and Euro do not teach measuring a mass spectrum.
However, Anderson teaches quantitative mass spectrometry measurement can be obtained as a ratio of (i) signal observed for one or more monitor peptides to (ii) the signal observed for the respective stable isotope labeled same-sequence internal standard SIS peptide added to the digest in known amount. The relative proportions may be used to calculate the hematocrit value of the blood sample (col.2, lines 59-65). Anderson teaches that peptide samples in the resulting eluate plate are analyzed with a system consisting of a 6490 triple quadropole mass spectrometer coupled to a 1290 Infinity UHPLC using a JetStream interface (col. 27, lines 13-16).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Abbott and Euro to measure a mass spectrum of a sample taught by Anderson, because Anderson teaches that the relative proportions of peptides and internal standard peptides could be used to calculate the hematocrit value of the blood sample. Each of Abbott and Anderson teach methods of determining hematocrit of blood samples. One of ordinary skill in the art would reasonably expect that replacing one known method of determining hematocrit with another would predictably result in the determination of hematocrit in a blood sample, and it was known in the art that mass spectrum measurements could be used to determine hematocrit in a blood sample.
Regarding claim 41-42 and 44, Abbott teaches freshly collected whole blood samples (col.3, Example 1). Euro teaches measuring NAD+:NADPH and NADP+:NADPH balances in animal tissues, cell and blood including whole blood (p.15, lines 16-17).
Abbott and Euro do not teach extracting a dried blood from a predetermined area of a dried blood spot on a blood collection material (claim 41), using a microneedle to obtain whole blood from an animal (claim 42), or wherein the predetermined area is about 5mm (claim 44).
Anderson teaches a measurement of hematocrit from a dried blood sample (abstract). Anderson teaches measuring proteins in complex biological samples including in clinical specimens such as human blood (in both liquid and dried forms) (description col. 1, lines 30-32). Anderson teaches a drop of fresh whole blood obtained by finger-prick or by venipuncture can be placed on filter paper, where it spreads into a circle and then dried to form a stable specimen, a “dried blood spot” (claim 42: the dried blood spot is obtained from using a microneedle to obtain whole blood from an animal) (col.11, line 55). Anderson teaches a disk 6mm in diameter is punched from the red area of dried blood using a standard hole punch; then the 6mm disk is placed in the bottom of a well of a flat bottomed 96-well plate, 20L of water is added and the plate is shaken for 30min to redissolve the DBS proteins (claim 41: extracting a dried blood from a predetermined area of dried blood spot on a blood collection material; claim 44: the area is about 5mm) (col. 25, lines 52-61). Anderson teaches that these methods provide important clinical information such as a complete blood count from a sample like a dried blood spot, in which no intact cells remain (col. 2, lines 16-18).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the method of using fresh whole blood taught by Abbott and Euro with the method of obtaining fresh blood using venipuncture to create a dried blood spot taught by Anderson to arrive at the claimed invention. Each of Abbott, Euro and Anderson teach measuring analytes in blood samples. One of ordinary skill in the art would have been motivated to use a dried blood spot taught by Anderson because Anderson teaches that a dried blood spot can be used to provide important clinical information such as complete blood count in a sample where no intact cells remain. There would have been a reasonable expectation of success since Anderson teaches measurement of hematocrit from a dried blood sample.
Regarding claims 73-76, Abbott teaches freshly collected whole blood samples (col.3, Example 1).
Euro teaches measuring NAD+:NADPH and NADP+:NADPH balances in animal tissues, cell and blood including whole blood (p.15, lines 16-17).
Abbott and Euro do not teach extracting a dried blood from a predetermined area of a dried blood spot on a blood collection material (claims 73 and 75).
Abbott and Euro are silent on using a microneedle to obtain whole blood from an animal (claims 74 and 76).
Anderson teaches a measurement of hematocrit from a dried blood sample (abstract). Anderson teaches measuring proteins in complex biological samples including in clinical specimens such as human blood (in both liquid and dried forms) (description col. 1, lines 30-32). Anderson teaches a drop of fresh whole blood obtained by finger-prick or by venipuncture can be placed on filter paper, where it spreads into a circle and then dried to form a stable specimen, a “dried blood spot” (claim 74: the dried blood spot is obtained from using a microneedle to obtain whole blood from an animal) (col.11, line 55). Anderson teaches a disk 6mm in diameter is punched from the red area of dried blood using a standard hole punch; then the 6mm disk is placed in the bottom of a well of a flat bottomed 96-well plate, 20L of water is added and the plate is shaken for 30min to redissolve the DBS proteins (claim 73: extracting a dried blood from a predetermined area of dried blood spot on a blood collection material) (col. 25, lines 52-61). Anderson teaches that these methods provide important clinical information such as a complete blood count from a sample like a dried blood spot, in which no intact cells remain (col. 2, lines 16-18).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the method of using fresh whole blood taught by Abbott and Euro with the method of obtaining fresh blood using venipuncture to create a dried blood spot taught by Anderson to arrive at the claimed invention. Each of Abbott, Euro and Anderson teach measuring analytes in blood samples. One of ordinary skill in the art would have been motivated to use a dried blood spot taught by Anderson because Anderson teaches that a dried blood spot can be used to provide important clinical information such as complete blood count in a sample where no intact cells remain.
Claims 15-17 and 31-32 are rejected under 35 U.S.C. 103 as being unpatentable over are rejected under 35 U.S.C. 103 as being unpatentable over Abbott et al. (US 4,699,887 published on October 13, 1987) in view of Euro et al. (WO 2022/008802 A1, published January 13, 2022) as applied to claim 7 above, and further in view of Haslam et al. (“Estimating the number of viable animal cells in multiwell culture—a tetrazolium-based assay”, Analytical Biochemistry, 2005, Vol. 336, Issue 2, pp.187-195).
The teachings of Abbott et al. and Euro et al. are discussed above.
Regarding claims 15 and 16, Abbott and Euro do not teach measuring a first absorption at the beginning of a predetermined reaction time and a second absorption at the conclusion of a predetermined reaction time.
However, Haslam teaches enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP results in reduced NADPH which reduces tetrazolium violet to its formazan (abstract). Haslam teaches the absorbencies can be measured at a series of time points to determine the course of the reaction (p.190, 1st column – GPD/INT assay protocol). Haslam further teaches measuring absorbencies at 0, 2, 5, 10, 15 and 20 minutes (i.e. predetermined reaction time) (p.190, Fig. 2). Haslam teaches that the absorbance values were plotted as functions of the number of cells per well and the time of incubation at a given cell density, and the results show that absorbance was proportional to the number of cells in the culture and the length of the GPD/INT assay (p.190, 2nd column GPD/INT assay of cells in culture).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Abbott and Euro to measure a first absorption and a second absorption at the beginning and conclusion of a predetermined reaction time taught by Haslam, because Haslam teaches the determination of absorption at pre-determined time points. Each of Abbott, Euro and Haslam teach measuring reaction products. One of ordinary skill in the art would have been motivated to measure a first absorption at the beginning of a predetermined time and a second absorption at the conclusion of the predetermined time to calculate a rate of formation of the redox product because Haslam teaches the absorbance values were proportional to the length of the GPD/INT assay. One of ordinary skill in the art would have found it beneficial to correlate the formation of NADPH with a standard curve to determine hematocrit of the blood sample.
Regarding claim 17, Abbott does not teach measuring absorbance at 350nm.
Euro teaches measuring change in absorbance of chromogen at an appropriate wavelength relating to the reagents used, such as wavelength 350nm (p.28, lines 17-18).
Haslam teaches measuring absorbencies at 0, 2, 5, 10, 15 and 20 minutes (i.e. predetermined reaction time) (p.190, Fig. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Abbott and Euro to measure absorbance at 350nm taught by Euro at predetermined timepoints taught by Haslam to arrive at the claimed invention. One of ordinary skill would have been motivated to select 350nm absorbance because Euro teaches measuring change in absorbance at an appropriate wavelength relating to the reagents used. One of ordinary skill would have found it beneficial to select 350nm as an appropriate wavelength based on the reagents used.
Regarding claim 31, Abbott and Euro do not teach measuring a first absorption at the beginning of a predetermined reaction time and a second absorption at the conclusion of a predetermined reaction time, or calculating a rate of formation of the redox product of the tetrazolium dye based on a change in absorption between the second absorption and the first absorption.
However, Haslam teaches an assay based on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in nonviable cells (abstract). Haslam teaches enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP results in reduced NADPH which reduces tetrazolium violet to its formazan (abstract). Haslam teaches the absorbencies can be measured at a series of time points to determine the course of the reaction (p.190, 1st column – GPD/INT assay protocol). Haslam further teaches measuring absorbencies at 0, 2, 5, 10, 15 and 20 minutes (i.e. predetermined reaction time) (p.190, Fig. 2). Haslam teaches that the absorbance values were plotted as functions of the number of cells per well and the time of incubation at a given cell density, and the results show that absorbance was proportional to the number of cells in the culture and the length of the GPD/INT assay (p.190, 2nd column GPD/INT assay of cells in culture).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Abbott and Euro to measure a first absorption and a second absorption at the beginning and conclusion of a predetermined reaction time taught by Haslam, because Haslam teaches the determination of absorption at pre-determined time points. Each of Abbott, Euro and Haslam teach measuring reaction products. One of ordinary skill in the art would have been motivated to measure a first absorption at the beginning of a predetermined time and a second absorption at the conclusion of the predetermined time to calculate a rate of formation of the redox product because Haslam teaches the absorbance values were proportional to the length of the GPD/INT assay. One of ordinary skill in the art would have found it beneficial to calculate formazan crystal formation and correlate the formation with a standard curve to correlate formazan formation with a desired sample metric.
Regarding claim 32, Abbott does not teach adding phenazine methosulfate to the lysed blood sample.
Euro teaches an electron carrier phenazine ethosulfate (PES) which receives an electron from an oxidized NAD metabolite and passes the electron to the final electron acceptor (e.g. tetrazolium bromide MTT) (p.20, lines 23-26).
Euro does not teach phenazine methosulfate.
Haslam teaches that in the presence of phenazine methosulfate (PMS) or phenazine ethosulfate (PES), the NADPH reduces iodonitrotetrazolium violet (INT) to its formazan (INT-F) (p.188, 2nd column, last paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Abbott and Euro to replace PES taught by Euro with PMS taught by Haslam, because Haslam teaches that phenazine methosulfate and phenazine ethosulfate are equivalent molecules. One of ordinary skill in the art would reasonably expect that replacing phenazine ethosulfate taught by Euro with phenazine methosulfate taught by Haslam would predictably result in the tetrazolium dye reducing to its formazan state, because it would amount to a simple substitution of one known electron carrier for another, and it was known in the art at the time of invention that both phenazine ethosulfate and phenazine methosulfate reduce tetrazolium dies to their formazan form.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over are rejected under 35 U.S.C. 103 as being unpatentable over Abbott et al. (US 4,699,887 published on October 13, 1987) in view of Euro et al. (WO 2022/008802 A1, published January 13, 2022) as applied to claim 7 above, and further in view of Gao et al. (“γ-6-phosphogluconolactone, a byproduct of the oxidative pentose phosphate pathway, contributes to AMPK activation through inhibition of PP2A”, Molecular Cell, 2019, Vol. 76, Issue 6, pp.857-871).
The teachings of Abbott et al. and Euro et al. are discussed above.
Regarding claim 20, Abbott and Euro do not teach measuring 6-phosphogluconolactone in the blood sample.
However, Gao teaches that glucose-6-phosphate dehydrogenase converts glucose-6-phosphate to 6-phosphogluconolactone (6-PGL) and produces NADPH (p.860, 1st column 1st paragraph; p.869 Figure 7G).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Abbott and Euro to measure the amount of 6-phosphogluconolactone taught by Gao, because Gao teaches that glucose-6-phosphate dehydrogenase converts glucose-6-phosphate to 6-PGL and produces NADPH. One of ordinary skill in the art would reasonably expect that measuring 6-PGL, a known reaction product of G6PDH enzyme would predictably result in an amount of 6-PGL that could be compared with a standard curve and used to determine the hematocrit of a blood sample, because it was known in the art at the time of invention that 6-PGL was produced by G6PDH enzyme along with NADPH.
Conclusion
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/DEEPA MISHRA/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657