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 .
Response to Amendment
The Amendment filed 08/10/2026 has been entered. Claims 16, 17, 20, 21, and 23-31 remain pending in the application. Claims 16, 30-31 have been amended, and no new claims have been added.
Status of Objections and Rejections
The objections to claims 16, 30, and 31 have been withdrawn in view of Applicant's amendment.
The rejection of claims 30-31 under 35 U.S.C. 101 is withdrawn in view of Applicant’s amendments.
New grounds of rejection under 35 U.S.C. 101 are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 112(b) are necessitated by the amendments.
New grounds of rejection under 35 U.S.C. 103 are necessitated by the amendments.
Response to Arguments
Applicant’s arguments, see pp. 12-14, filed 08/10/2026, with respect to the rejection of claims 16, 17, 20, 21, and 23-31 under 35 U.S.C. 101 and 35 U.S.C. 103 regarding the normalization coefficient, have been fully considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Applicant’s arguments, see p. 14, filed 08/10/2026, with respect to the rejection of claims 16, 17, 20, 21, and 23-31 under 35 U.S.C. 103 have been fully considered but they are not persuasive
Applicant argues (p.14) that both Beyette and Gentinetta omit the chemical dosing steps using (CO, KCN, sodium dithionite, and added heme), and therefore the claims are in condition for allowance.
The Examiner respectfully disagrees.
Claims 16, 30, and 31 state that the chemical dosing is selected from a list, and therefore teaching of each dosing is not required to meet the claim limitation as long as the content is determined for at least oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and hemopexin and bilirubin. References Beyette and Kuenstner satisfy determining the content of oxyhemoglobin, methemoglobin, carboxyhemoglobin, and bilirubin by spectral analysis. Gentinetta satisfies determining the content of heme bound to serum albumin and hemopexin using chemical dosing total hemopexin by adding heme. Although not required, Kuenstner ‘310 also teaches “some of the samples could be manipulated by the addition of sodium dithionite, carbon monoxide and/or potassium ferricyanide which would result in the production of deoxy-, carboxy- ,met-, and sulf-hemoglobin respectively” (col. 11, ll. 7-11). Therefore, the claim limitations have been satisfied.
Applicant argues (p.14) “Gentinetta does not disclose the second-derivative normalization coefficient required by claims 30 and 31…and does not teach the claimed iterative subtraction framework. The use of direct spectral deconvolution and extinction coefficients at fixed wavelengths, as disclosed in Gentinetta, is a materially different approach”.
The Examiner respectfully disagrees.
In response to applicant's argument that Gentinetta’s use of direct spectral deconvolution and extinction coefficients is materially different than the claimed second-derivative normalization coefficient, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Although Gentinetta determines heme-related components using spectral deconvolution and extinction coefficients, the reference is not relied upon for the claimed iterative subtraction framework. Rather, Beyette teaches this limitation and the removal of spectral components, while Gentinetta is relied upon for determining heme bound to serum albumin and hemopexin. Accordingly, Applicant improperly considers Gentinetta in isolation rather than the combined teachings of the references.
Claim Objections
Claims 16, 17, 30, and 31 are objected to because of the following informalities:
Regarding claim 16, lines 32 and 35 recite "with wavelength of the reference spectrum" which is grammatically unclear. Applicant may amend the claim to read “with respect to the wavelength of the reference spectrum” in order to clarify that wavelength is the variable with respect to which the second derivative is taken, without changing the scope of the claim.
Regarding claim 17, ll. 4-5, and 7 recite "with wavelength of the spectrum" which is grammatically unclear. Applicant may amend the claim to read “with respect to the wavelength of the spectrum” in order to clarify that wavelength is the variable with respect to which the second derivative is taken, without changing the scope of the claim.
Regarding claim 30, lines 31, 33-34 recite "with wavelength of the reference spectrum" which is grammatically unclear. Applicant may amend the claim to read “with respect to the wavelength of the reference spectrum” in order to clarify that wavelength is the variable with respect to which the second derivative is taken, without changing the scope of the claim.
Regarding claim 31, lines 33, 35-36 recite "with wavelength of the reference spectrum" which is grammatically unclear. Applicant may amend the claim to read “with respect to the wavelength of the reference spectrum” in order to clarify that wavelength is the variable with respect to which the second derivative is taken, without changing the scope of the claim.
Claim Rejections - 35 USC § 112
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 16, 17, 20, 21, and 23-31 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 16, second to last line recites “suitable” which is a relative term and renders the claim indefinite. The term is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Therefore, under broadest reasonable interpretation, the examiner understands the relative amount to encompass any treatment to the patient.
Claims 17, 20, 21, and 23-29 are rejected based on dependency of all of the limitations of claim 16.
Regarding claim 16, last two lines recite “get a transfusion for the subject in need thereof”. It does not clearly define the act required by the claimed method. It is unclear whether it requires administering a transfusion to the subject, causing or arranging for the subject to receive a transfusion, or the subject actually receiving the transfusion. Accordingly, the metes and bounds of the claimed method cannot be determined with reasonable certainty.
Claims 17, 20, 21, and 23-29 are rejected based on dependency of all of the limitations of claim 16.
Regarding claim 16, l. 5, step (a) recites “a wavelength of interest,” l. 14-15, step (c2) recite “the wavelength of interest associated to the first protein or heme-derived pigment,” and ll. 31-33, regarding the reference spectrum, state “over the wavelength of interest”. It is unclear whether the latter phrase refers to the wavelength range initially introduced in step (a) , or instead introduces a component specific wavelength range associated with the first protein or heme-derived pigment in step (c2). Accordingly, when the claim subsequently recites determining the second-derivative value “over the wavelength of interest,” it is unclear which wavelength range defines the scope of the calculation.
Claims 17, 20, 21, and 23-29 are rejected based on dependency of all of the limitations of claim 16.
Regarding claim 17, l. 5 and 7 recite “over the wavelength of interest”. Claim 16 from which claim 17 depends, l. 5, step (a) recites “a wavelength of interest” and l. 14-15, step (c) recite “the wavelength of interest associated to the first protein or heme-derived pigment”. It is unclear whether over the “wavelength of interest,” recited in claim 17 refers to the wavelength range initially introduced in step (a) , or instead introduces a component specific wavelength range associated with the first protein or heme-derived pigment in step (c2). Accordingly, when the claim subsequently recites determining the second-derivative value “over the wavelength of interest,” it is unclear which wavelength range defines the scope of the calculation.
Regarding claim 30, l. 5, step (a) recites “a wavelength of interest,” l. 14-15, step (c2) recite “the wavelength of interest associated to the first protein or heme-derived pigment,” and ll. 30-32, regarding the reference spectrum, state “over the wavelength of interest”. It is unclear whether the latter phrase refers to the wavelength range initially introduced in step (a) , or instead introduces a component specific wavelength range associated with the first protein or heme-derived pigment in step (c2). Accordingly, when the claim subsequently recites determining the second-derivative value “over the wavelength of interest,” it is unclear which wavelength range defines the scope of the calculation.
Regarding claim 31, l. 6, step (a) recites “a wavelength of interest,” l. 15-16, step (c2) recite “the wavelength of interest associated to the first protein or heme-derived pigment,” and ll. 31-33, regarding the reference spectrum, state “over the wavelength of interest”. It is unclear whether the latter phrase refers to the wavelength range initially introduced in step (a) , or instead introduces a component specific wavelength range associated with the first protein or heme-derived pigment in step (c2). Accordingly, when the claim subsequently recites determining the second-derivative value “over the wavelength of interest,” it is unclear which wavelength range defines the scope of the calculation.
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 16, 17, 20, 21, and 23-29 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (Step 2A/1)(i.e., a law of nature, a natural phenomenon, or an abstract idea) without practical application (Step 2A/2) or significantly more (Step 2B) (See MPEP 2106). Although the instant claims encompass a process (claims 16, 26-27, and 29)(Step 1), they are directed to the following judicial exceptions including abstract ideas through mental processes, mathematical calculations and law of nature through natural correlation (Step 2, Prong 1):
Claim 16 recites:
- a) measuring an initial spectrum of the sample on a wavelength range of interest,
- b) setting a current spectrum that is iteratively updated at step d) as the initial spectrum,.
- c) iterating the following steps:
- c1) c1) determining the content of a first protein or heme-derived pigment in the sample independently of a content of a second protein or heme-derived pigment, wherein the first and second proteins or heme-derived pigments are selected among oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin, heme bound to hemopexin and bilirubin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing,
- c2) deducing an overall spectral component on the wavelength range of interest associated to the first protein or heme-derived pigment based on the determined content for the first protein or heme-derived pigment, and
- c3) removing the deduced spectral component from the current spectrum,
- d) outputting the determined contents in the first protein or heme-derived pigment, wherein each of steps c1), c2), and c3) is performed for the first protein or heme-derived pigment, and step c) is iterated successively until the content of the first protein or heme-derived pigment is determined for at least oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and hemopexin and bilirubin,
wherein each of steps c1), c2), and c3) is performed for the first protein or heme-derived pigment, and step c) is iterated successively until the content of the first protein or heme-derived pigment is determined for at least oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and hemopexin and bilirubin,
wherein said the spectral analysis comprises:
providing a reference spectrum associated with the first protein or heme- derived pigment, and
calculating a normalization coefficient between the current spectrum and the reference spectrum, the normalization coefficient is the ratio between the value of second derivative of the first protein or heme-derived pigment in the current spectrum and the value of second derivative of the first protein or heme derived pigment in the reference spectrum, the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative of the second derivative with wavelength of the reference spectrum; and
wherein the chemical dosing is selected from the group consisting of: dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof.
Claims 26-27, and 29 recite predicting if a subject is at risk for a disorder, diagnosing a disorder, defining the stages of a disorder, qualifying or disqualifying a medical bag, and monitoring treatment of a disorder.
Step 2A/1: The claims are directed to the concept of analyzing a biological sample to determine protein content by measuring and processing spectral data.
Claim 16, Recitation b) is directed to the abstract idea of a mental process in naming a spectrum which can be performed in the mind. Recitation c1) is directed to the abstract idea of mathematical calculations of spectral data. Providing a reference spectrum is retrieval of data by remembering what a known protein’s spectrum looks like. A human with training could mentally compare the shape of two graphs (the current spectrum and the reference spectrum). Calculating the normalization coefficient is a simple ratio (value of the second derivative from the sample divided by the value of the second derivative of a protein peak from the reference). Ratios are basic arithmetic operations that can be carried out mentally, on paper, or with pencil and paper, without requiring a computer or specialized machine. A derivative is a simple rate of change, and a second derivative is the rate of change of the first derivative. Labeling different points on the curve and calculating these rates and maximums can be performed in the mind and by hand. Conceptually, a person could look at curves of different spectra, estimate which spectrum has the sharpest slope change, and proceed in order by mentally ranking the graphs from largest to smallest change. Recitation c2) is a mathematical operation that applies the normalization coefficient of c1) to the reference spectrum in order to produce another dataset. Such mathematical manipulation of data is an abstract idea. Because the step can be performed in the human mind or with pen and paper (by scaling and subtracting known spectral curves), it falls within the judicial exception of a mental process. Recitation c3) can be performed by overlaying the deduced overall spectrum of c2) with the current spectrum and tracing the residual curve that is created. Since it can be carried out mentally or with simple arithmetic tools, it falls into the mental process abstract idea category along with recitation d). Outputting is conceptually no different from mentally reporting results, and a human can easily write the numbers down after calculating them from the previous step. As a whole, the method depends on the naturally occurring relationship between protein concentration and spectral absorbance, which is a law of nature through natural correlation (Mayo Collaborative Services v. Prometheus Laboratories, Inc., 566 U.S. 66 (2012). The claim is therefore directed toward a judicial exception.
Additionally, for claims 26-27, and 29, predicting if a subject is at risk for a disorder, diagnosing a disorder, defining the stages of a disorder, qualifying or disqualifying a medical bag, and monitoring treatment of a disorder can be performed in the mind by comparing the output of protein content in the sample to known values. The claims are therefore directed toward a judicial exception.
Step 2A/2: These judicial exceptions are not integrated into a practical application.
Measuring an initial spectrum without specifying even generic instrumentation is merely data gathering (pre-solution activity, see MPEP 2106.05 (g)) only for the purpose of executing the abstract idea of the determination step. Simply outputting the result of a mental process by displaying the results of the determination step of protein content is considered post-solution activity (MPEP 2106.05 (g)). . Applicant has amended the claims to recite step “e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof”. However, MPEP 2106.04(d)(2) states that “in order to qualify as a "treatment" or "prophylaxis"…the claim limitation in question must affirmatively recite an action that effects a particular treatment or prophylaxis for a disease or medical condition”(Emphasis Added). An example from MPEP 2106.04(d)(2) that satisfies this requirement is the case of Vanda which claims “(1) performing a genotyping assay to determine if a patient has a genotype associated with poor drug metabolism; and (2) administering iloperidone to the patient in a dose range that depends on the patient’s genotype”. Here, the medical condition is poor drug metabolism and it is a particular treatment in that it includes a dose range and depends on the patient’s genotype. The instant amended claims only recite administering hydroxyurea and chelators iron “for the subject in need thereof,” which does not specify which disease or medical condition is being treated nor how much. Additionally, “get a transfusion” creates ambiguity as to who is performing what affirmative step and can be interpreted as arranging for or obtaining a transfusion rather than performing or administering it. For example, MPEP 2106.04(d)(2) states that "prescribing a topical steroid to a patient with eczema" is not a positive limitation because it does not require that the steroid actually be used by or on the patient. “Get a transfusion” is therefore not a practical application of the abstract idea. Furthermore, the amended claim recites “any other suitable molecule” which also does not transform the judicial exception into an abstract idea. MPEP 2106.04(d)(2) states "administering a suitable medication to a patient." This administration step is not particular, and is instead merely instructions to "apply" the exception in a generic way. Thus, the administration step does not integrate the mental analysis step into a practical application”. Therefore, claim 16 fails to provide a practical application of the judicial exceptions from Step 2A/1.
Additionally, for claims 26-27, and 29, predicting if a subject is at risk for a disorder, diagnosing a disorder, defining the stages of a disorder, qualifying or disqualifying a medical bag, and monitoring treatment of a disorder can be performed in the mind by comparing the output of protein content in the sample to known values. Once the comparisons are made, there is no further action. Accordingly, these additional elements do not integrate the abstract idea into a practical application because it does not impose any meaningful limits on practicing the abstract ideas. The claims are directed to an abstract idea with no practical application.
Step 2B: The claims do not include additional elements that are sufficient to amount to
significantly more than the judicial exception
Claims 16: The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception. As mentioned in Step 2A/2, the previous process steps of the claims are abstract ideas, mere data gathering, and are routine and conventional in the art. The claims fail to provide a specific improvement to a computer, technology, or a technical field resulting in a practical application of the judicial exception. There is no specific instrumentation (e.g. spectrophotometer) required in claim 16 to provide the initial spectrum, reference spectrum, or means for determining a protein content of the sample.
The use of a ratio of second derivatives for normalization in spectral analysis is well-understood, routine and conventional in the art. A reference Myers (US 6473632 B1; 2002) measures the concentration of hemoglobin in blood samples by using a scaling factor created from a ratio of a second derivative value of a sample spectrum and a second derivative value at a reference wavelength (col. 4, ll. 66-67-col. 5, l. 1; Fig. 9). Myers also references Kuenstner (US 5377674 A; 1995, see 35 U.S.C. 103 rejection below) as teaching a similar methodology (col. 1, ll. 26-36; Myers)(column 7, lines 15-18; Kuenstner). These references establish that such mathematical derivative manipulation in regard to blood protein quantification was well-established. Likewise, iterative quantification of individual species in a mixture using reference spectrum subtraction with scaling coefficients was also well-known, routine and conventional before the effective filing date of the instant application. A reference, Yaghoobi et al. (“Fast Sparse Raman Spectral Unmixing for Chemical Fingerprinting and Quantification”; 2016), teaches “The algorithm is based on iteratively subtracting the contribution of selected spectra and updating the contribution of each spectrum…The iteration terminates when the maximum number of expected chemicals has been found” (p. 99950E-1, para. 2). Reference Beyette applies a similar iteration to protein samples ([0013])(See 35 U.S.C. 103 rejection below). Finally, using chemical dosing of a sample (using CO, KCN, etc.) to quantify different blood protein species via spectroscopy is well-known, routine and conventional in the art (See claims 23-24 of reference Rieders et al., (US6194218B1; 2001) as well as Beyette and Kuestner ‘674 in the 35 U.S.C. 103 rejection below. Accordingly, the claim limitations of iterative spectral subtraction, second-derivative ratio normalization, and chemical dosing of a sample do not amount to significantly more than the judicial exception, and the rejection of claim 16 under 35 U.S.C. 101 is maintained. Claims 17, 20-21, 23-29 are rejected upon dependency of all the limitations of claim 16.
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 16, 17, 20, 21, 25, 26, and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Beyette et al. (US 20060034730 A1) in view of Garcia et al. (“A contribution to the derivative ratio spectrum method”), Kuenstner (US 6393310 B1 and Kuestner US 5377674 A, fully incorporated by reference), as evidenced by Norris et al. (“Optimization of Mathematical Treatments of Raw Near-Infrared Signal in the Measurement of Protein in Hard Red Spring Wheat. I. Influence of Particle Size”), Agilent (“Uses of Derivative Spectroscopy”), Gentinetta (US 20200237652 A1), and UNICEF et al. (“Neonatal care clinical guidelines”).
Regarding claim 16, Beyette teaches a method of treatment of heme-related or hemoprotein-related disorder in a subject (“These sequential samples may be used to assess severity of the condition or efficacy of treatments,” for “the problem of differentially diagnosing a subarachnoid hemorrhage from a traumatic spinal tap,” wherein the concentration of bilirubin depends on the extent of aneurysm rupture; [0081], [0099], [0101]),
wherein a content of proteins or heme-derived pigment in a biological sample of a subject is measured (“obtaining a cerebrospinal fluid sample from an individual,” and “determining concentrations of analytes in the cerebrospinal fluid sample,” wherein the analytes are “methemoglobin, oxyhemoglobin, bilirubin”; [0012], Abstract; [0064]), the method of treatment comprising the steps of:
measuring an initial spectrum of the sample on a wavelength range of interest ((e) obtaining a sample of the fluid and generating a measured spectral absorption curve (F) of the sample over each wavelength range determined in step (d); [0067])("Spectrophotometer results of CSF following SAH obtained from LP" shown in Fig. 1 includes a wavelength),
b) setting a current spectrum that is iteratively updated at step d) as the initial spectrum (Paragraph [0067] of Beyette gives a broad overview of the iteration steps establishing that the iteration begins with the measured sample spectrum, while paragraph [0098] explains that after subtracting the bilirubin curve, it forms a new residual curve R(n), and states that S(n)=R(n), wherein S(n) is the measured spectral absorption curve or initial spectrum. Beyette then states, “the new residual is then used in place of S(n) in the algorithm as steps (1)-(5) are repeated”. The residual curve is therefore the new initial spectrum and therefore the current spectrum),
c) iterating the following steps:
c1) determining the content of a first protein (methemoglobin) or heme-derived pigment in the sample independently of a content of a second protein or heme-derived pigment (bilirubin), wherein the first and second proteins or heme-derived pigments are selected among oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin, heme bound to hemopexin and bilirubin (Beyette teaches this limitation in paragraph [0098] by independently determining the concentrations of bilirubin and methemoglobin through comparison of the residual spectrum (current) with the standard bilirubin and methemoglobin spectra. Paragraph [0098] determines bilirubin concentration by selecting Bi(n), the standard bilirubin curve corresponding to a particular concentration that is closest to the residual. The same method is applied to independently determine the concentration of methemoglobin),
the determination being carried out by using a spectral analysis of the current spectrum ("These standard curves are used as quantization levels for estimating the bilirubin or methemoglobin values in a CSF/SAH sample"; [0098]) or/and a chemical dosing,
c2) deducing an overall spectral component on the wavelength range of interest associated to the first protein or heme-derived pigment based on the determined content for the first protein or heme-derived pigment (Steps (a)-(b) of paragraph [0067] state a standard curve is obtained for the contaminant (first protein, methemoglobin) and determines the wavelength where it dominates. Interpolative curves (g) at different concentrations for the first protein are created from the standard curve, and a curve is deduced to (h) select which standard contaminant spectral absorption curve of the contaminant is closest in distance to F.sub.sc over the wavelength range where the contaminant dominates), and
c3) removing the deduced spectral component from the current spectrum (steps (i) and (k) explain that residual curves are created for each of the contaminant and analyte after the closest standard curves are deduced, respectively. Paragraph [0098] provides the equations Rm(n) = |Sinit(n)-Mi(n), showing the subtraction of the deduced standard curve from the current spectrum/residual curve); [0067]),
d) outputting the determined contents in the first protein or heme-derived pigment, wherein each of steps c1), c2), and c3) is performed for the first protein or heme-derived pigment, and step c) is iterated successively (“iteratively repeating steps (h)-(k) until a standard contaminant curve and a standard analyte curve are selected in consecutive iterations,” wherein the algorithm “yields an estimated value of the concentration of the analyte”; [0067], [0016]) until the content of the first protein or heme-derived pigment is determined for at least oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and hemopexin and bilirubin ("The iterative procedure is continued until M.sub.i and B.sub.i converge," for the result of "estimating bilirubin and methemoglobin in the SAH data," wherein M.sub.i and B.sub.i are the absorption curves of methemoglobin and bilirubin, respectively with an index of "i" iterations; [0098])(See Figs. 8a, 8b, 9,14-19).
wherein the spectral analysis comprises:
providing a reference spectrum associated with the first protein or heme-derived pigment ((b) using a spectrophotometer to generate at least one standard spectral absorption curve for the contaminant; [0067]), and
calculating a normalization coefficient between the current spectrum and the reference spectrum (Beyette teaches calculating scaling factors for normalizing a measured spectral absorption curve relative to pre-measured standard spectral absorption curves (See paras. [0067, [0089]-[0090]).
Modified Beyette fails to teach:
the normalization coefficient is the ratio between the value of second derivative of the first protein or heme-derived pigment in the current spectrum and the value of second derivative of the first protein or heme derived pigment in the reference spectrum, the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum; and
Determining the content of oxyhemoglobin, carboxyhemoglobin, heme bound to serum albumin and heme bound to hemopexin by using spectral analysis or/and chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof.
Beyette instead teaches using a scaling factor that is based on the standard curves (reference spectrum) to scale the measured spectrum ([0067]) and also states that the concentration of the proteins can be determined using a second derivative mathematical algorithm ([0064]).
Garcia teaches the normalization coefficient is the ratio between a value of a current spectrum and the value of a reference spectrum (“spectrum obtained as the ratio of that for the mixture and a standard spectrum for one of its components,” wherein there are many values within each spectrum; pp. 83-84).
Garcia is reasonably pertinent to the problem of using a reference spectrum to normalize a multicomponent mixture spectrum for individual component quantitation through derivative ratios. Beyette analyzes spectra with overlapping contributions from methemoglobin and bilirubin and uses standard spectra from each component to determine the respective concentrations in the form of a scaling factor ([0067]). Rote addresses this same analytical struggle for component overlap in teaching the derivative ratio spectrum method as a solution by dividing a binary mixture spectrum by a standard spectrum with the use of second derivatives (p. 84, col. 1, paras. 2-3; p. 91, col. 2, para. 3, last l.). Garcia notes the benefits of this method in that it “facilitates optimization of the working conditions and diminishes quantitation errors” (p. 84, para. 2). Beyette even states that a second derivative algorithm can be used in combination with the modified minimum distance algorithm used in step (h) for selecting the appropriate standard curve to apply to each iterated residual/current spectrum ([0064], [0067]). This provides evidence for a reasonable expectation of success if Garcia’s derivative analysis were applied to the iterated spectra. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette to incorporate the teachings of Garcia by applying a reference spectrum ratio technique to the measured, residual, and standard absorption curves already taught by Beyette because this would improve the quantitative resolution of individual components in the overlapping spectrum, and this involves applying a known technique to a known method ready for improvement to yield predictable results (See MPEP 2143(I)(D)).
Modified Beyette fails to teach the normalization coefficient is the ratio between the value of second derivative of the first protein or heme-derived pigment in the current spectrum and the value of second derivative of the first protein or heme derived pigment in the reference spectrum, the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum;
Determining the content of oxyhemoglobin, carboxyhemoglobin, heme bound to serum albumin, and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof.
Kuenstner teaches the normalization coefficient is the ratio between the value of second derivative of a protein in a current spectrum and the value of second derivative in the reference spectrum (The spectra would be normalized by dividing by the area of the absolute value of the first or second derivative spectrum in the region of interest; column 3, lines 27-28)(Kuenstner ‘674 teaches that “the second derivative of absorbance at an analyte wavelength of 1744 nm divided by the second derivative of absorbance at a reference wavelength of 1674 nm” which calculates “the patient's hemoglobin concentration is 6.6 g/dL”; column 7, lines 15-18), and
Determining the content of oxyhemoglobin and carboxyhemoglobin by using a spectral analysis (measuring hemoglobin species concentrations including, oxyhemoglobin…carboxyhemoglobin, methemoglobin; column 1 lines 19-21).
Kuenstner is considered to be analogous to the claimed invention because it is in the same field of endeavor for spectrophotometric determination of hemoglobin concentration using spectral analysis. Modified Beyette explains that “second-derivative spectra gave rise to even smaller errors,” (Garcia, p. 91, col. 1, ll. 2-3) and that “The derivative ratio spectrum method allows the resolution of binary mixtures of analytes with satisfactory results that are further improved by the use of normalized spectra as divisor spectra and spectral derivative orders above one” (Garcia, p. 93, ll. 6-12). It is known that use of second derivative normalization has many benefits as expressed by Norris et al. (“Optimization of Mathematical Treatments of Raw Near-Infrared Signal in the Measurement of Protein in Hard Red Spring Wheat. I. Influence of Particle Size”; 1994). Norris states that “Normalization of data…such that second derivative divided by second derivative at optimum wavelengths gave an average bias error of-0.02% with a standard deviation of bias of 0.12% …in predicting protein content of samples” (Abstract). Finally, Modified Beyette already contemplates the use of second derivative analysis by stating that a second derivative algorithm can be used in combination with the modified minimum distance algorithm used in step (h) for selecting the appropriate standard curve to apply to each iterated residual/current spectrum (Beyette, [0064], [0067]). This provides evidence that applying a second derivative calculation to the current spectrum would yield predictable results. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia to incorporate the teachings of Kuenstner by applying a known ratio of second derivatives as a normalization coefficient to the current spectrum because this would obtain a measure of the individual proteins that reduces unwanted spectral variation and quantitation errors. This involves applying a known technique to a known method ready for improvement (See MPEP 2143(I)(D)).
Modified Beyette fails to teach the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum;
Determining the content of heme bound to serum albumin and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof.
Agilent teaches the value of second derivative being defined as the maximum value over a wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum (“For the even order derivatives there is a peak maximum or minimum at the same λmax as the absorbance spectrum…Taking the difference between the highest maximum and the lowest minimum gives the best signal-to noise ratio,” wherein this calculation would naturally yield the absolute value of the difference between a minima and a maxima since the maximum is always larger than the minimum; p. 3, col. 1, para. 3; See second equation in col. 3 for “the second-derivative determination”).
Agilent is considered to be analogous to the claimed invention because it is in the same field of endeavor for spectrophotometric quantification of multicomponent samples using derivative spectral techniques (Title, p. 5, col. 1, para. 2, ll. 6-13). Both Modified Beyette and Agilent teach the use of second derivative analysis to quantify individual components within a sample mixture, with modified Beyette stating that “higher values of SNR may be considered analogous to better estimation of bilirubin” (Beyette, [0103]). Agilent states that defining the second derivative value to be the difference between the highest maximum and the lowest minimum gives the best signal-to noise ratio, which (p. 3, col. 1, para. 3). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia, Kuenstner, and Norris to incorporate the teachings of Agilent by defining the value of the ratio of second derivatives which includes the reference spectrum to be an absolute value of the difference between a minima and a maxima of the second derivative because it would improve the signal-to-noise ratio, and this involves applying a known technique to a known method ready for improvement (See MPEP 2143(I)(D)).
Modified Beyette fails to teach:
Determining the content of heme bound to serum albumin and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof.
Gentinetta teaches:
Determining the content of heme bound to serum albumin and heme bound to hemopexin (the different spectral characteristics of heme when it is bound to albumin or to hemopexin can be used to determine the concentration of the heme-hemopexin complex in the solution; [0169]) via chemical dosing by
dosing total hemopexin by adding heme (met-Hb (Fe.sup.3+) (15 μM in PBS) or hemin bound to human albumin (25 μM in PBS) was incubated with 10 μM human Hpx,” wherein under broadest reasonable interpretation dosing is achieved since a reagent (met-Hb or hemin bound to human albumin) is added that delivers heme to Hpx, revealing Hpx content via spectral analysis (“in order to follow the transition of met-Hb/hemin to heme-Hpx over time”); [0193])(the concentrations of met-Hb/hemin and heme-Hpx in the reaction mixtures were resolved by deconvolution of the full spectrum; [0193]) ,
dosing total hemopexin by adding heme and sodium dithionite, and
dosing total hemopexin by adding heme, CO and sodium dithionite
Gentinetta is considered to be analogous to the claimed invention because it is in the same field of endeavor for determining the protein content in a biological sample. Heme bound to serum albumin and heme bound to hemopexin are natural, clinically relevant heme-protein complexes that interfere spectrally in blood. Modified Beyette states that “a potential pitfall in the clinical usefulness of a bilirubin assay to detect an SAH is the interference caused by hemoglobin concentrations” (Beyette, [0077]). A person of ordinary skill in the art would have been motivated to configure Modified Beyette’s normalization framework to quantify them for the same reasons it quantifies hemoglobin derivatives (Kuenstner, Abstract), they are spectroscopically distinguishable and clinically important, and the extension would have been a predictable use of the same methods. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia, Kuenstner, Norris, and Agilent to incorporate the teachings of Gentinetta by determining the content of heme-HSA and heme-HPX and subtracting these peaks from the current spectrum because this would reduce interference when trying to obtain the bilirubin concentration, and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Modified Beyette fails to teach:
e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof.
However, Modified Beyette does teach applying the method to patient cerebral spinal fluid samples in order to assess severity of the condition (such as ruptured aneurysm) or efficacy of treatments ([0081],[0006]).
UNICEF teaches e) administering a treatment with hydroxyurea, chelators iron or any other suitable molecule or get a transfusion for the subject in need thereof (Had newborn jaundice with bilirubin levels high enough to require a blood transfusion; p. 22, ll. 4-5; See bilirubin concentrations corresponding to “Exchange transfusion guidelines” on p. 71, Fig. 9).
UNICEF is reasonably pertinent to the problem of determining bilirubin content in a biological sample and using the value to guide treatment. Beyette teaches that rapid and sensitive CSF analysis is important for directing patient care in suspected SAH and that sequential CSF samples may be used to assess condition severity or treatment efficacy ([0006],[0081]). Similarly, UNICEF addresses CSF in connection with hemorrhage and further teaches using quantitatively determined bilirubin levels to guide therapeutic intervention such as a blood transfusion. The Neonatal Care Clinical Guidelines were developed in cooperation with UNICEF and WHO to provide standardized, high quality clinical care based on evidence. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia, Kuenstner, Norris, Agilent, and Gentinetta to incorporate the teachings of UNICEF by suggesting treatment of a transfusion based on a patient’s bilirubin content because it would improve clinical management of a hemorrhage related condition, and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Regarding claim 17, Modified Beyette teaches the method for determining according to claim 16, wherein step c) is iterated by decreasing values of second derivatives of the proteins (Paragraph [0066] of Beyette states “the algorithm provides better estimate of bilirubin in SAH than methemoglobin since the bilirubin spectrum provides higher SNR over the 450 nm to 500 nm wavelength region at all concentration levels,” in which case bilirubin is considered the signal and methemoglobin the noise. Hence, the noise of methemoglobin is subtracted from the spectrum first leaving the bilirubin wherein the second derivative is used to calculate the SNR).
Regarding claim 20, Modified Beyette teaches the method for determining according to claim 16, wherein at least one of the following properties is fulfilled:
a spectral analysis is carried out for the oxyhemoglobin and the normalization coefficient is calculated for at least one wavelength chosen in three intervals, the first interval encompassing wavelengths comprised between 415 nanometers and 417 nanometers, the second interval encompassing wavelengths comprised between 542 nanometers and 544 nanometers and the third interval encompassing wavelengths comprised between 576 nanometers and 578 nanometers, the interval being preferably the third interval;
a spectral analysis is carried out for the methemoglobin and the normalization coefficient is calculated for at least one wavelength chosen in the range of 350 nanometers to 750, and
a spectral analysis is carried out for the bilirubin and the normalization coefficient is calculated for at least one wavelength chosen in the range of 350 nanometers to 750 nanometers nanometers (linear combination of pre-measured bilirubin and methemoglobin spectra over the wavelengths of significance, i.e., between 390 and 420 nm and 450 to 600 nm. Therefore, over a certain set of wavelengths in the spectral data, we adjust the SAH curve with a scale factor; [0089]; Beyette; Figs. 12-13).
Regarding claim 21, Modified Beyette teaches the method for determining according to claim 16, wherein a spectral analysis is carried out for the plasma heme and the normalization coefficient is calculated for at least one wavelength chosen in the range of 300 nanometers to 750 nanometers using reference spectra (In CSF analysis, the wavelength range from 350 to 600 nm is scanned to obtain an entire range of spectra for a specific concentration; Beyette, [0058]).
Regarding claim 25, Modified Beyette teaches The method for determining according to claim 16, wherein when the contents in oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and bilirubin are successively determined, at least one of oxyhemoglobin, methemoglobin (“potassium ferricyanide,” wherein the addition of KCN will naturally reveal methemoglobin; Kuenstner ‘310, column 11, lines 6-8), carboxyhemoglobin, heme bound to serum albumin content and bilirubin is determined by a chemical dosing.
Regarding claim 26, Modified Beyette teaches the method for determining according to claim 16, wherein the biological sample is from a subject and wherein the method further comprises the steps of:
predicting that the subject is at risk of suffering from the heme-related or hemoprotein- related disorder on the determined contents in protein; or
diagnosing the heme-related or hemoprotein-related disorder based on the determined contents in protein (“analysis of human spinal fluid may be diagnostic of… neurodegeneration,” wherein neurodegeneration is a hemeprotein-related disorder; Beyette, [0059]); or
defining the stages of the heme-related or hemoprotein-related disorder based on the determined contents in protein.
Regarding claim 29, Modified Beyette teaches the method for determining according to claim 16, wherein the biological sample is from a subject suffering from a heme-related or hemoprotein-related disorder and having received a treatment against said heme-related or hemoprotein- related disorder (In order to "assess severity of the condition or efficacy of treatments," wherein the condition is the hemoprotein-related condition of "neurodegeneration," the patient would needed to have been initially treated; Beyette, [0081][0059]), the method further comprising the step of:
monitoring the determined contents in protein with monitoring the treatment against heme- related or hemoprotein-related disorder in a subject suffering from the heme- related or hemoprotein-related disorder and having received the treatment (The method of assessing a condition in an individual may further comprise the step of obtaining sequential cerebrospinal fluid samples from the individual. These sequential samples may be used to assess severity of the condition or efficacy of treatments"; Beyette, [0081], claim 24).
Claims 23-24, 27-28 are rejected under 35 U.S.C. 103 as being unpatentable over Beyette et al. (US 20060034730 A1) in view of Garcia et al. (“A contribution to the derivative ratio spectrum method”), Kuenstner (US 6393310 B1 and Kuestner US 5377674 A, fully incorporated by reference), as evidenced by Norris et al. (“Optimization of Mathematical Treatments of Raw Near-Infrared Signal in the Measurement of Protein in Hard Red Spring Wheat. I. Influence of Particle Size”), Agilent (“Uses of Derivative Spectroscopy”), Gentinetta (US 20200237652 A1), and UNICEF et al. (“Neonatal care clinical guidelines”), as applied to claim 16 above, and in further view of Samsoondar et al. (US 20050019936 A1).
Regarding claim 23, Modified Beyette teaches the method for determining according to claim 16,
Modified Beyette fails to teach wherein at the step for outputting, the determined contents comprise additional contents in protein which are different from the content in oxyhemoglobin, from the content in methemoglobin, from the content in heme bound to serum albumin and hemopexin from the content in bilirubin, the additional contents being the contents of proteins selected from the group consisting of: carboxylated hemoglobin, myoglobin, porphyrins, porphobilinogen, urobilin, products of catabolism of heme by heme oxygenase, and products of bilirubin degradation and oxidation products of organ dysfunction and cytosis, metabolic substrates, and metabolic degradation products
Samsoondar teaches biliverdin (biliverdin (BV); [0006]).
Samsoondar is considered to be analogous to the claimed invention because it is in the same field of endeavor for determining the protein content in a biological sample. The art of Beyette already teaches determining the concentration of bilirubin in a SAH CSF blood sample for the benefit of diagnosing the condition of a patient (Abstract). Biliverdin is a direct and well-known degradation product of bilirubin through the action of biliverdin reductase. Measuring biliverdin in addition to bilirubin and the other proteins provides predictable, incremental diagnostic value without requiring a change in the underlying spectrophotometric methods taught by the combined arts. Beyette already teaches applying derivative spectroscopy to distinguish between overlapping bilirubin and hemoglobin spectra. One of ordinary skill in the art would reasonably expect to apply the same analytical techniques to biliverdin, since it is another chromophore with characteristic absorption peaks in the visible spectrum. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the protein analysis method taught by Beyette in view of Garcia, Kuenstner, Norris, Agilent, Gentinetta, and UNICEF to incorporate the teachings of Samsoondar by including biliverdin as an analyte to be quantified because “bile pigments bilirubin (BR) and biliverdin (BV) are typical blood components”(Samsoondar; [0006]), and this involves combining prior art elements according to known methods to yield predictable results ((See MPEP 2143(I)(A)).
Regarding claim 24, Modified Beyette teaches the method for determining according to claim 23, wherein the products of bilirubin degradation and oxidation are selected from the group consisting of biliverdin (biliverdin (BV); Samsoondar, [0006]) and heme boxes.
Regarding claim 27, Modified Beyette teaches the method for determining according to claim 16,
Modified Beyette fails to teach the biological sample is from a medical bag, and wherein the method further comprises the step of qualifying or disqualifying the medical bag based on the determined contents in protein.
Modified Beyette does teach "to reduce error readings, only the absorption at peak wavelengths are considered" ([0058]).
Samsoondar teaches wherein the biological sample is from a medical bag (“plasma specimens contained in a blood bag”, where a blood bag is a form of medical bag; paragraph 27, line 2), and wherein the method further comprises the step of qualifying or disqualifying the medical bag based on the determined contents in protein (on the basis of the results from measurement of any one or more of these interferents at a time, in comparison with reference measurements of various levels of interferents, a decision is made concerning whether to reject or accept the plasma contained in the blood bag; paragraph 28, lines 12-15)
Samsoondar is considered to be analogous to the claimed invention because it is in the same field of endeavor for determining the protein content in a biological sample. Ultimately, the goal of reducing error readings to objectively quantify spectral output when analyzing blood in the CSF to assess a condition, will be fulfilled by screening medical bags for interferences (Beyette, [0058][0008]). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the protein analysis method taught by Beyette in view of Garcia, Kuenstner, Norris, Agilent, Gentinetta, and UNICEF to incorporate the teachings of Samsoondar to incorporate the teachings of Samsoondar by choosing the biological sample to be from a medical bag, determining its contents in protein and deciding to qualifying or disqualifying the medical bag based on the determined contents in protein because it would help to “determine if a plasma specimen representative of plasma in a blood bag contains interferents and if so, to what extent”; (Samsoondar; Abstract, lines 3-4), and this involves combining prior art elements according to known methods to yield predictable results ((See MPEP 2143(I)(A)).
Regarding claim 28, Modified Beyette in view of Samsoondar teaches the method for determining according to claim 27, wherein the medical bag is a blood bag (“plasma specimens contained in a blood bag; Samsoondar paragraph 27, line 2).
Claim 30 is rejected under 35 U.S.C. 103 as being unpatentable over Beyette et al. (US 20060034730 A1) in view of Garcia et al. (“A contribution to the derivative ratio spectrum method”), Kuenstner (US 6393310 B1 and Kuestner US 5377674 A, fully incorporated by reference), as evidenced by Norris et al. (“Optimization of Mathematical Treatments of Raw Near-Infrared Signal in the Measurement of Protein in Hard Red Spring Wheat. I. Influence of Particle Size”), Agilent (“Uses of Derivative Spectroscopy”), and Gentinetta (US 20200237652 A1).
Regarding claim 30, Beyette teaches a non-transitory computer readable medium (a processor; [0016]), storing instructions that, (subjects the output signal to an algorithm; [0016]), the computer program being loadable into a data-processing unit (the algorithm is functionally capable of performing the intended use of being loadable into a data-processing unit) when executed by a processor cause the processor to perform steps of a method for determining a content of proteins or heme-derived pigment in a biological sample, ("spectral content of the CSF obtained from LP," wherein "the analytes in the CSF sample to be analyzed comprise methemoglobin, oxyhemoglobin, bilirubin, or a combination thereof"; [0064])(Beyette defines CSP and LP to be cerebral spinal fluid and lumbar puncture, respectively), the method for determining comprising the steps of:
measuring an initial spectrum of the sample on a wavelength range of interest ((e) obtaining a sample of the fluid and generating a measured spectral absorption curve (F) of the sample over each wavelength range determined in step (d); [0067])("Spectrophotometer results of CSF following SAH obtained from LP" shown in Fig. 1 includes a wavelength),
b) setting a current spectrum that is iteratively updated at step d) as the initial spectrum (Paragraph [0067] of Beyette gives a broad overview of the iteration steps establishing that the iteration begins with the measured sample spectrum, while paragraph [0098] explains that after subtracting the bilirubin curve, it forms a new residual curve R(n), and states that S(n)=R(n), wherein S(n) is the measured spectral absorption curve or initial spectrum. Beyette then states, “the new residual is then used in place of S(n) in the algorithm as steps (1)-(5) are repeated”. The residual curve is therefore the new initial spectrum and therefore the current spectrum),
c) iterating the following steps:
c1) determining the content of a first protein (methemoglobin) or heme-derived pigment in the sample independently of a content of a second protein or heme-derived pigment (bilirubin), wherein the first and second proteins or heme-derived pigments are selected among oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin, heme bound to hemopexin and bilirubin (Beyette teaches this limitation in paragraph [0098] by independently determining the concentrations of bilirubin and methemoglobin through comparison of the residual spectrum (current) with the standard bilirubin and methemoglobin spectra. Paragraph [0098] determines bilirubin concentration by selecting Bi(n), the standard bilirubin curve corresponding to a particular concentration that is closest to the residual. The same method is applied to independently determine the concentration of methemoglobin),
the determination being carried out by using a spectral analysis of the current spectrum ("These standard curves are used as quantization levels for estimating the bilirubin or methemoglobin values in a CSF/SAH sample"; [0098]) or/and a chemical dosing,
c2) deducing an overall spectral component on the wavelength range of interest associated to the first protein or heme-derived pigment based on the determined content for the first protein or heme-derived pigment (Steps (a)-(b) of paragraph [0067] state a standard curve is obtained for the contaminant (first protein, methemoglobin) and determines the wavelength where it dominates. Interpolative curves (g) at different concentrations for the first protein are created from the standard curve, and a curve is deduced to (h) select which standard contaminant spectral absorption curve of the contaminant is closest in distance to F.sub.sc over the wavelength range where the contaminant dominates), and
c3) removing the deduced spectral component from the current spectrum (steps (i) and (k) explain that residual curves are created for each of the contaminant and analyte after the closest standard curves are deduced, respectively. Paragraph [0098] provides the equations Rm(n) = |Sinit(n)-Mi(n), showing the subtraction of the deduced standard curve from the current spectrum/residual curve); [0067]),
d) outputting the determined contents in the first protein or heme-derived pigment, wherein each of steps c1), c2), and c3) is performed for the first protein or heme-derived pigment, and step c) is iterated successively (“iteratively repeating steps (h)-(k) until a standard contaminant curve and a standard analyte curve are selected in consecutive iterations,” wherein the algorithm “yields an estimated value of the concentration of the analyte”; [0067], [0016]) until the content of the first protein or heme-derived pigment is determined for at least oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and hemopexin and bilirubin ("The iterative procedure is continued until M.sub.i and B.sub.i converge," for the result of "estimating bilirubin and methemoglobin in the SAH data," wherein M.sub.i and B.sub.i are the absorption curves of methemoglobin and bilirubin, respectively with an index of "i" iterations; [0098])(See Figs. 8a, 8b, 9,14-19).
wherein the spectral analysis comprises:
providing a reference spectrum associated with the first protein or heme-derived pigment ((b) using a spectrophotometer to generate at least one standard spectral absorption curve for the contaminant; [0067]), and
calculating a normalization coefficient between the current spectrum and the reference spectrum (Beyette teaches calculating scaling factors for normalizing a measured spectral absorption curve relative to pre-measured standard spectral absorption curves (See paras. [0067, [0089]-[0090]).
Modified Beyette fails to teach:
the normalization coefficient is the ratio between the value of second derivative of the first protein or heme-derived pigment in the current spectrum and the value of second derivative of the first protein or heme derived pigment in the reference spectrum, the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum; and
Determining the content of oxyhemoglobin, carboxyhemoglobin, heme bound to serum albumin and heme bound to hemopexin by using spectral analysis or/and chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Beyette instead teaches using a scaling factor that is based on the standard curves (reference spectrum) to scale the measured spectrum ([0067]) and also states that the concentration of the proteins can be determined using a second derivative mathematical algorithm ([0064]).
Garcia teaches the normalization coefficient is the ratio between a value of a current spectrum and the value of a reference spectrum (“spectrum obtained as the ratio of that for the mixture and a standard spectrum for one of its components,” wherein there are many values within each spectrum; pp. 83-84).
Garcia is reasonably pertinent to the problem of using a reference spectrum to normalize a multicomponent mixture spectrum for individual component quantitation through derivative ratios. Beyette analyzes spectra with overlapping contributions from methemoglobin and bilirubin and uses standard spectra from each component to determine the respective concentrations in the form of a scaling factor ([0067]). Rote addresses this same analytical struggle for component overlap in teaching the derivative ratio spectrum method as a solution by dividing a binary mixture spectrum by a standard spectrum with the use of second derivatives (p. 84, col. 1, paras. 2-3; p. 91, col. 2, para. 3, last l.). Garcia notes the benefits of this method in that it “facilitates optimization of the working conditions and diminishes quantitation errors” (p. 84, para. 2). Beyette even states that a second derivative algorithm can be used in combination with the modified minimum distance algorithm used in step (h) for selecting the appropriate standard curve to apply to each iterated residual/current spectrum ([0064], [0067]). This provides evidence for a reasonable expectation of success if Garcia’s derivative analysis were applied to the iterated spectra. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette to incorporate the teachings of Garcia by applying a reference spectrum ratio technique to the measured, residual, and standard absorption curves already taught by Beyette because this would improve the quantitative resolution of individual components in the overlapping spectrum, and this involves applying a known technique to a known method ready for improvement to yield predictable results (See MPEP 2143(I)(D)).
Modified Beyette fails to teach the normalization coefficient is the ratio between the value of second derivative of the first protein or heme-derived pigment in the current spectrum and the value of second derivative of the first protein or heme derived pigment in the reference spectrum, the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum;
Determining the content of oxyhemoglobin, carboxyhemoglobin, heme bound to serum albumin, and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Kuenstner teaches the normalization coefficient is the ratio between the value of second derivative of a protein in a current spectrum and the value of second derivative in the reference spectrum (The spectra would be normalized by dividing by the area of the absolute value of the first or second derivative spectrum in the region of interest; column 3, lines 27-28)(Kuenstner ‘674 teaches that “the second derivative of absorbance at an analyte wavelength of 1744 nm divided by the second derivative of absorbance at a reference wavelength of 1674 nm” which calculates “the patient's hemoglobin concentration is 6.6 g/dL”; column 7, lines 15-18), and
Determining the content of oxyhemoglobin and carboxyhemoglobin by using a spectral analysis (measuring hemoglobin species concentrations including, oxyhemoglobin…carboxyhemoglobin, methemoglobin; column 1 lines 19-21).
Kuenstner is considered to be analogous to the claimed invention because it is in the same field of endeavor for spectrophotometric determination of hemoglobin concentration using spectral analysis. Modified Beyette explains that “second-derivative spectra gave rise to even smaller errors,” (Garcia, p. 91, col. 1, ll. 2-3) and that “The derivative ratio spectrum method allows the resolution of binary mixtures of analytes with satisfactory results that are further improved by the use of normalized spectra as divisor spectra and spectral derivative orders above one” (Garcia, p. 93, ll. 6-12). It is known that use of second derivative normalization has many benefits as expressed by Norris et al. (“Optimization of Mathematical Treatments of Raw Near-Infrared Signal in the Measurement of Protein in Hard Red Spring Wheat. I. Influence of Particle Size”; 1994). Norris states that “Normalization of data…such that second derivative divided by second derivative at optimum wavelengths gave an average bias error of-0.02% with a standard deviation of bias of 0.12% …in predicting protein content of samples” (Abstract). Finally, Modified Beyette already contemplates the use of second derivative analysis by stating that a second derivative algorithm can be used in combination with the modified minimum distance algorithm used in step (h) for selecting the appropriate standard curve to apply to each iterated residual/current spectrum (Beyette, [0064], [0067]). This provides evidence that applying a second derivative calculation to the current spectrum would yield predictable results. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia to incorporate the teachings of Kuenstner by applying a known ratio of second derivatives as a normalization coefficient to the current spectrum because this would obtain a measure of the individual proteins that reduces unwanted spectral variation and quantitation errors. This involves applying a known technique to a known method ready for improvement (See MPEP 2143(I)(D)).
Modified Beyette fails to teach the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum;
Determining the content of heme bound to serum albumin and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Agilent teaches the value of second derivative being defined as the maximum value over a wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum (“For the even order derivatives there is a peak maximum or minimum at the same λmax as the absorbance spectrum…Taking the difference between the highest maximum and the lowest minimum gives the best signal-to noise ratio,” wherein this calculation would naturally yield the absolute value of the difference between a minima and a maxima since the maximum is always larger than the minimum; p. 3, col. 1, para. 3; See second equation in col. 3 for “the second-derivative determination”).
Agilent is considered to be analogous to the claimed invention because it is in the same field of endeavor for spectrophotometric quantification of multicomponent samples using derivative spectral techniques (Title, p. 5, col. 1, para. 2, ll. 6-13). Both Modified Beyette and Agilent teach the use of second derivative analysis to quantify individual components within a sample mixture, with modified Beyette stating that “higher values of SNR may be considered analogous to better estimation of bilirubin” (Beyette, [0103]). Agilent states that defining the second derivative value to be the difference between the highest maximum and the lowest minimum gives the best signal-to noise ratio, which (p. 3, col. 1, para. 3). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia, Kuenstner, and Norris to incorporate the teachings of Agilent by defining the value of the ratio of second derivatives which includes the reference spectrum to be an absolute value of the difference between a minima and a maxima of the second derivative because it would improve the signal-to-noise ratio, and this involves applying a known technique to a known method ready for improvement (See MPEP 2143(I)(D)).
Modified Beyette fails to teach:
Determining the content of heme bound to serum albumin and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Gentinetta teaches:
Determining the content of heme bound to serum albumin and heme bound to hemopexin (the different spectral characteristics of heme when it is bound to albumin or to hemopexin can be used to determine the concentration of the heme-hemopexin complex in the solution; [0169]) via chemical dosing by
dosing total hemopexin by adding heme (met-Hb (Fe.sup.3+) (15 μM in PBS) or hemin bound to human albumin (25 μM in PBS) was incubated with 10 μM human Hpx,” wherein under broadest reasonable interpretation dosing is achieved since a reagent (met-Hb or hemin bound to human albumin) is added that delivers heme to Hpx, revealing Hpx content via spectral analysis (“in order to follow the transition of met-Hb/hemin to heme-Hpx over time”); [0193])(the concentrations of met-Hb/hemin and heme-Hpx in the reaction mixtures were resolved by deconvolution of the full spectrum; [0193]) ,
dosing total hemopexin by adding heme and sodium dithionite, and
dosing total hemopexin by adding heme, CO and sodium dithionite
Gentinetta is considered to be analogous to the claimed invention because it is in the same field of endeavor for determining the protein content in a biological sample. Heme bound to serum albumin and heme bound to hemopexin are natural, clinically relevant heme-protein complexes that interfere spectrally in blood. Modified Beyette states that “a potential pitfall in the clinical usefulness of a bilirubin assay to detect an SAH is the interference caused by hemoglobin concentrations” (Beyette, [0077]). A person of ordinary skill in the art would have been motivated to configured Modified Beyette’s normalization framework to quantify them for the same reasons it quantifies hemoglobin derivatives (Kuenstner, Abstract), they are spectroscopically distinguishable and clinically important, and the extension would have been a predictable use of the same methods. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia, Kuenstner, Norris, and Agilent to incorporate the teachings of Gentinetta by determining the content of heme-HSA and heme-HPX and subtracting these peaks from the current spectrum because this would reduce interference when trying to obtain the bilirubin concentration, and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Beyette et al. (US 20060034730 A1) in view of Garcia et al. (“A contribution to the derivative ratio spectrum method”), Kuenstner (US 6393310 B1 and Kuestner US 5377674 A, fully incorporated by reference), as evidenced by Norris et al. (“Optimization of Mathematical Treatments of Raw Near-Infrared Signal in the Measurement of Protein in Hard Red Spring Wheat. I. Influence of Particle Size”), Agilent (“Uses of Derivative Spectroscopy”), and Gentinetta (US 20200237652 A1).
Regarding claim 31, Beyette teaches an apparatus for determining a content of proteins or heme- derived pigment in a biological sample ("spectral content of the CSF obtained from LP," wherein "the analytes in the CSF sample to be analyzed comprise methemoglobin, oxyhemoglobin, bilirubin, or a combination thereof"; [0064])(Beyette defines CSP and LP to be cerebral spinal fluid and lumbar puncture, respectively), wherein the apparatus at least comprises
a spectrophotometer (A spectrophotometer; [0067]), dosing materials ("A 22 gauge spinal needle,", wherein under broadest reasonable interpretation, the examiner understands a dosing material to be any solid, fluid, or gas that can be used in the process of dosing; [0131]) and an analysis system (a processor which subjects the output signal to an algorithm which yields an estimated value of the concentration of the analyte; [0016]), the apparatus being adapted to carry out a method for determining the content of protein or heme-derived pigment in a biological sample, the method for determining comprising the steps of:
measuring an initial spectrum of the sample on a wavelength range of interest ((e) obtaining a sample of the fluid and generating a measured spectral absorption curve (F) of the sample over each wavelength range determined in step (d); [0067])("Spectrophotometer results of CSF following SAH obtained from LP" shown in Fig. 1 includes a wavelength),
b) setting a current spectrum that is iteratively updated at step d) as the initial spectrum (Paragraph [0067] of Beyette gives a broad overview of the iteration steps establishing that the iteration begins with the measured sample spectrum, while paragraph [0098] explains that after subtracting the bilirubin curve, it forms a new residual curve R(n), and states that S(n)=R(n), wherein S(n) is the measured spectral absorption curve or initial spectrum. Beyette then states “the new residual is then used in place of S(n) in the algorithm as steps (1)-(5) are repeated”. The residual curve is therefore the new initial spectrum and therefore the current spectrum),
c) iterating the following steps:
c1) determining the content of a first protein (methemoglobin) or heme-derived pigment in the sample independently of a content of a second protein or heme-derived pigment (bilirubin), wherein the first and second proteins or heme-derived pigments are selected among oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin, heme bound to hemopexin and bilirubin (Beyette teaches this limitation in paragraph [0098] by independently determining the concentrations of bilirubin and methemoglobin through comparison of the residual spectrum (current) with the standard bilirubin and methemoglobin spectra. Paragraph [0098] determines bilirubin concentration by selecting Bi(n), the standard bilirubin curve corresponding to a particular concentration that is closest to the residual. The same method is applied to independently determine the concentration of methemoglobin),
the determination being carried out by using a spectral analysis of the current spectrum ("These standard curves are used as quantization levels for estimating the bilirubin or methemoglobin values in a CSF/SAH sample"; [0098]) or/and a chemical dosing,
c2) deducing an overall spectral component on the wavelength range of interest associated to the first protein or heme-derived pigment based on the determined content for the first protein or heme-derived pigment (Steps (a)-(b) of paragraph [0067] state a standard curve is obtained for the contaminant (first protein, methemoglobin) and determines the wavelength where it dominates. Interpolative curves (g) at different concentrations for the first protein are created from the standard curve, and a curve is deduced to (h) select which standard contaminant spectral absorption curve of the contaminant is closest in distance to F.sub.sc over the wavelength range where the contaminant dominates), and
c3) removing the deduced spectral component from the current spectrum (steps (i) and (k) explain that residual curves are created for each of the contaminant and analyte after the closest standard curves are deduced, respectively. Paragraph [0098] provides the equations Rm(n) = |Sinit(n)-Mi(n), showing the subtraction of the deduced standard curve from the current spectrum/residual curve); [0067]),
d) outputting the determined contents in the first protein or heme-derived pigment, wherein each of steps c1), c2), and c3) is performed for the first protein or heme-derived pigment, and step c) is iterated successively (“iteratively repeating steps (h)-(k) until a standard contaminant curve and a standard analyte curve are selected in consecutive iterations,” wherein the algorithm “yields an estimated value of the concentration of the analyte”; [0067], [0016]) until the content of the first protein or heme-derived pigment is determined for at least oxyhemoglobin, methemoglobin, carboxyhemoglobin, heme bound to serum albumin and hemopexin and bilirubin ("The iterative procedure is continued until M.sub.i and B.sub.i converge," for the result of "estimating bilirubin and methemoglobin in the SAH data," wherein M.sub.i and B.sub.i are the absorption curves of methemoglobin and bilirubin, respectively with an index of "i" iterations; [0098])(See Figs. 8a, 8b, 9,14-19).
wherein the spectral analysis comprises:
providing a reference spectrum associated with the first protein or heme-derived pigment ((b) using a spectrophotometer to generate at least one standard spectral absorption curve for the contaminant; [0067]), and
calculating a normalization coefficient between the current spectrum and the reference spectrum (Beyette teaches calculating scaling factors for normalizing a measured spectral absorption curve relative to pre-measured standard spectral absorption curves (See paras. [0067, [0089]-[0090]).
Modified Beyette fails to teach:
the normalization coefficient is the ratio between the value of second derivative of the first protein or heme-derived pigment in the current spectrum and the value of second derivative of the first protein or heme derived pigment in the reference spectrum, the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum; and
Determining the content of oxyhemoglobin, carboxyhemoglobin, heme bound to serum albumin and heme bound to hemopexin by using spectral analysis or/and chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Beyette instead teaches using a scaling factor that is based on the standard curves (reference spectrum) to scale the measured spectrum ([0067]) and also states that the concentration of the proteins can be determined using a second derivative mathematical algorithm ([0064]).
Garcia teaches the normalization coefficient is the ratio between a value of a current spectrum and the value of a reference spectrum (“spectrum obtained as the ratio of that for the mixture and a standard spectrum for one of its components,” wherein there are many values within each spectrum; pp. 83-84).
Garcia is reasonably pertinent to the problem of using a reference spectrum to normalize a multicomponent mixture spectrum for individual component quantitation through derivative ratios. Beyette analyzes spectra with overlapping contributions from methemoglobin and bilirubin and uses standard spectra from each component to determine the respective concentrations in the form of a scaling factor ([0067]). Rote addresses this same analytical struggle for component overlap in teaching the derivative ratio spectrum method as a solution by dividing a binary mixture spectrum by a standard spectrum with the use of second derivatives (p. 84, col. 1, paras. 2-3; p. 91, col. 2, para. 3, last l.). Garcia notes the benefits of this method in that it “facilitates optimization of the working conditions and diminishes quantitation errors” (p. 84, para. 2). Beyette even states that a second derivative algorithm can be used in combination with the modified minimum distance algorithm used in step (h) for selecting the appropriate standard curve to apply to each iterated residual/current spectrum ([0064], [0067]). This provides evidence for a reasonable expectation of success if Garcia’s derivative analysis were applied to the iterated spectra. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette to incorporate the teachings of Garcia by applying a reference spectrum ratio technique to the measured, residual, and standard absorption curves already taught by Beyette because this would improve the quantitative resolution of individual components in the overlapping spectrum, and this involves applying a known technique to a known method ready for improvement to yield predictable results (See MPEP 2143(I)(D)).
Modified Beyette fails to teach the normalization coefficient is the ratio between the value of second derivative of the first protein or heme-derived pigment in the current spectrum and the value of second derivative of the first protein or heme derived pigment in the reference spectrum, the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum;
Determining the content of oxyhemoglobin, carboxyhemoglobin, heme bound to serum albumin, and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Kuenstner teaches the normalization coefficient is the ratio between the value of second derivative of a protein in a current spectrum and the value of second derivative in the reference spectrum (The spectra would be normalized by dividing by the area of the absolute value of the first or second derivative spectrum in the region of interest; column 3, lines 27-28)(Kuenstner ‘674 teaches that “the second derivative of absorbance at an analyte wavelength of 1744 nm divided by the second derivative of absorbance at a reference wavelength of 1674 nm” which calculates “the patient's hemoglobin concentration is 6.6 g/dL”; column 7, lines 15-18), and
Determining the content of oxyhemoglobin and carboxyhemoglobin by using a spectral analysis (measuring hemoglobin species concentrations including, oxyhemoglobin…carboxyhemoglobin, methemoglobin; column 1 lines 19-21).
Kuenstner is considered to be analogous to the claimed invention because it is in the same field of endeavor for spectrophotometric determination of hemoglobin concentration using spectral analysis.
Modified Beyette explains that “second-derivative spectra gave rise to even smaller errors,” (Garcia, p. 91, col. 1, ll. 2-3) and that “The derivative ratio spectrum method allows the resolution of binary mixtures of analytes with satisfactory results that are further improved by the use of normalized spectra as divisor spectra and spectral derivative orders above one” (Garcia, p. 93, ll. 6-12). It is known that use of second derivative normalization has many benefits as expressed by Norris et al. (“Optimization of Mathematical Treatments of Raw Near-Infrared Signal in the Measurement of Protein in Hard Red Spring Wheat. I. Influence of Particle Size”; 1994). Norris states that “Normalization of data…such that second derivative divided by second derivative at optimum wavelengths gave an average bias error of-0.02% with a standard deviation of bias of 0.12% …in predicting protein content of samples” (Abstract). Finally, Modified Beyette already contemplates the use of second derivative analysis by stating that a second derivative algorithm can be used in combination with the modified minimum distance algorithm used in step (h) for selecting the appropriate standard curve to apply to each iterated residual/current spectrum (Beyette, [0064], [0067]). This provides evidence that applying a second derivative calculation to the current spectrum would yield predictable results. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia to incorporate the teachings of Kuenstner by applying a known ratio of second derivatives as a normalization coefficient to the current spectrum because this would obtain a measure of the individual proteins that reduces unwanted spectral variation and quantitation errors. This involves applying a known technique to a known method ready for improvement (See MPEP 2143(I)(D)).
Modified Beyette fails to teach the value of second derivative of the reference spectrum being defined as the maximum amplitude over the wavelength range of interest of the second derivative with wavelength of the reference spectrum or the maximum value over the wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum;
Determining the content of heme bound to serum albumin and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Agilent teaches the value of second derivative being defined as the maximum value over a wavelength range of interest of an absolute value of the difference between a minima and a maxima of the second derivative with wavelength of the reference spectrum (“For the even order derivatives there is a peak maximum or minimum at the same λmax as the absorbance spectrum…Taking the difference between the highest maximum and the lowest minimum gives the best signal-to noise ratio,” wherein this calculation would naturally yield the absolute value of the difference between a minima and a maxima since the maximum is always larger than the minimum; p. 3, col. 1, para. 3; See second equation in col. 3 for “the second-derivative determination”).
Agilent is considered to be analogous to the claimed invention because it is in the same field of endeavor for spectrophotometric quantification of multicomponent samples using derivative spectral techniques (Title, p. 5, col. 1, para. 2, ll. 6-13). Both Modified Beyette and Agilent teach the use of second derivative analysis to quantify individual components within a sample mixture, with modified Beyette stating that “higher values of SNR may be considered analogous to better estimation of bilirubin” (Beyette, [0103]). Agilent states that defining the second derivative value to be the difference between the highest maximum and the lowest minimum gives the best signal-to noise ratio, which (p. 3, col. 1, para. 3). Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia, Kuenstner, and Norris to incorporate the teachings of Agilent by defining the value of the ratio of second derivatives which includes the reference spectrum to be an absolute value of the difference between a minima and a maxima of the second derivative because it would improve the signal-to-noise ratio, and this involves applying a known technique to a known method ready for improvement (See MPEP 2143(I)(D)).
Modified Beyette fails to teach:
Determining the content of heme bound to serum albumin and heme bound to hemopexin, the determination being carried out by using a spectral analysis of the current spectrum or/and a chemical dosing, wherein the chemical dosing is selected from the group consisting of:
dosing oxyhemoglobin by adding CO, dosing methemoglobin by adding KCN, dosing plasma heme by adding CO and sodium dithionite, dosing heme bound to hemopexin by adding sodium dithionite dosing total hemopexin by adding heme, dosing total hemopexin by adding heme and sodium dithionite, and dosing total hemopexin by adding heme, CO and sodium dithionite; and
Gentinetta teaches:
Determining the content of heme bound to serum albumin and heme bound to hemopexin (the different spectral characteristics of heme when it is bound to albumin or to hemopexin can be used to determine the concentration of the heme-hemopexin complex in the solution; [0169]) via chemical dosing by
dosing total hemopexin by adding heme (met-Hb (Fe.sup.3+) (15 μM in PBS) or hemin bound to human albumin (25 μM in PBS) was incubated with 10 μM human Hpx,” wherein under broadest reasonable interpretation dosing is achieved since a reagent (met-Hb or hemin bound to human albumin) is added that delivers heme to Hpx, revealing Hpx content via spectral analysis (“in order to follow the transition of met-Hb/hemin to heme-Hpx over time”); [0193])(the concentrations of met-Hb/hemin and heme-Hpx in the reaction mixtures were resolved by deconvolution of the full spectrum; [0193]) ,
dosing total hemopexin by adding heme and sodium dithionite, and
dosing total hemopexin by adding heme, CO and sodium dithionite
Gentinetta is considered to be analogous to the claimed invention because it is in the same field of endeavor for determining the protein content in a biological sample. Heme bound to serum albumin and heme bound to hemopexin are natural, clinically relevant heme-protein complexes that interfere spectrally in blood. Modified Beyette states that “a potential pitfall in the clinical usefulness of a bilirubin assay to detect an SAH is the interference caused by hemoglobin concentrations” (Beyette, [0077]). A person of ordinary skill in the art would have been motivated to configured Modified Beyette’s normalization framework to quantify them for the same reasons it quantifies hemoglobin derivatives (Kuenstner, Abstract), they are spectroscopically distinguishable and clinically important, and the extension would have been a predictable use of the same methods. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the teachings of Beyette in view of Garcia, Kuenstner, Norris, and Agilent to incorporate the teachings of Gentinetta by determining the content of heme-HSA and heme-HPX and subtracting these peaks from the current spectrum because this would reduce interference when trying to obtain the bilirubin concentration, and this involves combining prior art elements according to known methods to yield predictable results (See MPEP 2143(I)(A)).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Rote et al., 2009 (instant PTO-892), teaches the ratio derivative method by dividing the spectrum of a mixture by the standardized spectra of each analyte.
Petibois, et al., 2009 (instant PTO-892), teaches spectral subtraction iteration similar to Beyette.
No claims are allowed.
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/V.S./Examiner, Art Unit 1758
/MARIS R KESSEL/Supervisory Patent Examiner, Art Unit 1758
