DETAILED ACTION
CONTINUED EXAMINATION UNDER 37 CFR 1.114 AFTER FINAL REJECTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant’s submission of RCE and the amendment filed on April 30, 2026 have been entered. The claims pending in this application are claims 15, 17, 19-28, 37, 38, and 43-48 wherein claims 21, 22, and 37 have been withdrawn due to the restriction requirement mailed on July 23, 2014. The rejection not reiterated from the previous office action is hereby withdrawn in view of applicant’s amendment filed on April 30, 2026. Claims 15, 17, 19, 20, 23-28, 38, and 43-48 will be examined.
Claim Objections
Claim 15 is objected to because of the following informality: the phrase “on which none of steps (1) to (6) is performed” in the end of the claim should be deleted.
Appropriate correction is required.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 15, 17, 19, 20, 23-28, 38, and 43-46 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Xiang et al., (US 2006/0040283 A1, published on February 23, 2006) in view of Lenz et al., (US 2010/0173295 A1), Cory et al., (US 2006/ 0090214A1, published on April 27, 2006), Chu (US Patent No. 6,291,180 B1, published on September 18, 2001), Espina et al., (US 2013/0137094 A1, priority date: January 25, 2010) and Kennedy et al., (US 2010/0131432 A1, priority date: November 17, 2008).
Regarding claims 15, 17, 20, 23-28, and 43, Xiang et al., teach (1) mounting at least one part of the biological sample (ie., supraoptic nucleus (SON) from brain) on at least one support (ie., the glass slide), (2) optionally treating the at least one part of the biological sample with a sample treatment agent (eg., ethanol or xylene), wherein said treatment can be carried out at any stage of the procedure before step (6), (3) staining the at least one part of the fixed biological sample mounted on the at least one support with a first cell or tissue staining agent (ie., stained with anti-oxytocin-neurophysin antibody), (4) determining target cells or tissue comprising biomolecules of interest by observing the at least one part stained in step (3) (ie., red color stained by anti-oxytocin-neurophysin antibody), (5) separating the target cells or tissue (ie., the tissue stained by anti-oxytocin-neurophysin antibody) comprising biomolecules of interest (ie., RNA) from the remainder of the biological sample mounted on the at least one support, and (6) isolating the biomolecules from the target cells or tissue separated in step (5) wherein the biomolecules comprise RNA as recited in claim 15, the method comprises step (2) and the sample treatment agent is a de-waxing agent (ie., xylene) as recited in claim 17 wherein the de-waxing agent is a de-paraffinizing agent such as xylene as recited in claims 20 and 23 (see page 33, paragraph [0375]), the at least one support (ie., the glass slide) is transparent as recited in claim 24, the at least one support is made of glass (ie., the glass slide) or a transparent polymer as recited in claim 25, the target cells or tissue comprising biomolecules of interest from the biological sample in step (4) is observed under light or backlight (eg., by using Leica DM RX microscope) as recited in claim 26, the target cells or tissue comprising biomolecules of interest from the biological sample in step (4) is observed under a microscope (eg., Leica DM RX microscope) as recited in claim 27, the target cells or tissue is/are separated in step (5) by microdissection, laser capture microdissection, scraping, or cutting out as recited in claim 28, and the biological sample comprises at least one section of 0.5 to 12 µm (ie., 8 µm) thickness of a non-formalin-fixed paraffin-embedded sample as recited in claim 43 (see pages 32-36, Example 14, paragraphs [0368] to [390]).
Regarding claim 38, Xiang et al., teach that the fixed biological sample is divided in sections of 0.5 to 12 µm thickness (ie., 8µm), and in step (1), the at least one of the support is a transparent support, in step (2) the at least one part of the biological sample is treated with the sample treatment agent and the sample treatment agent is a de-waxing agent (ie., xylene), and in step (4), the target cells or tissue is/are determined in the stained sample section (see pages 32-36, Example 14, paragraphs [0368] to [390]). Although Xiang et al., do not teach that, in step (5) of claim 15, the remainder of the fixed sample (ie., fixed sections stained by anti-oxytocin-neurophysin antibody) is discarded, in view of the teachings of Xiang et al., it is obvious to one having ordinary skill in the art at the time the invention was made to discard the slides after scraping fixed sections stained by anti-oxytocin-neurophysin antibody from the slides into Eppendorf tubes (see paragraph [0371]).
Xiang et al., do not disclose that the biological sample is fixed with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition, and a wax embedded and staining the at least one part of the biological sample mounted on the at least one support for a time period that is not more than 60 seconds with a first cell or tissue staining agent and rinsing the at least one part of the biological sample with water wherein the non-crosslinking composition does not comprise formaldehyde and the isolated RNA has an RNA Integrity Number (RIN) value comparable to a reference RIN value, and the reference RIN value is the RIN value of RNA that is isolated from the biological sample as recited in claim 15, the fixed biological sample is non-formalin-fixed paraffin-embedded sample (non-FFPE- sample) as recited in claim 19, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for a time period that is less than 45 seconds with the first cell or tissue staining agent as recited in claim 44, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for a time period that is less than 30 seconds with the first cell or tissue staining agent as recited in claim 45, and step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for a time period that is less than 15 seconds with the first cell or tissue staining agent as recited in claim 46.
Lenz et al., teach that a biological sample is fixed with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition, and wax embedded (see abstract, Examples 8 and 9, and claims 1-21).
Regarding claims 15 and 19, since Cory et al., teach that brain tissue fixed in Bouin’s solution for 5 hr are embedded in paraffin, and 8 µm sections are transferred to silane-coated microscope slides and stained with hematoxylin and eosin (see pages 6 and 7, paragraph [0098]), Cory et al., disclose that the fixed biological sample is a wax embedded sample as recited in claim 15 and the fixed biological sample is non-formalin-fixed paraffin-embedded sample (non-FFPE- sample) as recited in claim 19.
Regarding claims 15, 38, and 44-46, since Chu teaches that “[D]eparaffinized tissue section slides which are in slide holders are placed vertically into a staining dish with 500 mL of hematoxylin solution for 10 seconds with ultrasound followed by washing with running tap water in a staining dish for 5 seconds with ultrasound. The slides are placed in 95% ethyl alcohol for 5 seconds with ultrasound and counterstained in eosin-phloxine solution for 10
seconds with ultrasound” (see column 18), Chu discloses staining the at least one part of the biological sample mounted on the at least one support for a time period that is not more than 60 seconds with a first cell or tissue staining agent and rinsing the at least one part of the biological sample with water as recited in claim 15 wherein said section is stained for not more than 60 seconds with a first tissue staining agent and rinsed said section with water as recited in claim 38, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for a time period that is less than 45 seconds with the first cell or tissue staining agent as recited in claim 44, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for a time period that is less than 30 seconds with the at least one cell or tissue staining agent as recited in claim 45, and step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for a time period that is less than 15 seconds with the at least one cell or tissue staining agent as recited in claim 46.
Espina et al., teach that preservation of RNA in a tissue is evaluated by calculating the RNA integrity number (RIN) of a RNA and the RIN of the RNA fixed by their method is compared with the RIN of the RNA preserved by a commercially available, room temperature RNA preservation method, “RNALater®”, and the RIN of an untreated RNA (see paragraphs [0014], [0044] and [0045] and Figure 10).
Kennedy et al., teach that RNA quality can be measured by a calculated RIN and “[A]dvantages of RIN include obtain a numerical assessment of the integrity of RNA; directly comparing RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs; and ensuring repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used” (see paragraph [0111]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the methods as recited in claims 15, 19, 38, and 44-46 by staining the at least one part of the biological sample mounted on the at least one support for a time period that is less than 15 seconds with a first cell or tissue staining agent and rinsing the at least one support with water wherein the biological sample is fixed with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition and a wax embedded, the non-crosslinking composition does not comprise formaldehyde, and is not Methacarn solution, the fixed biological sample is non-formalin-fixed paraffin-embedded sample (non-FFPE-sample such as DSP-fixed paraffin-embedded biological sample), the isolated RNA has an RNA Integrity Number (RIN) value comparable to a reference RIN value, and the reference RIN value is the RIN value of RNA that is isolated from the biological sample in view of the prior arts of Xiang et al., Lenz et al., Cory et al., Chu, Espina et al., and Kennedy et al.. One having ordinary skill in the art would have been motivated to do so because Lenz et al., have successfully fixed a biological sample with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition, and wax embedded (see abstract, Examples 8 and 9, and claims 1-21), and provided advantages of their composition and the method “[T]he present compositions and method provide samples which are treated in a way that isolation of most of the biological components originally contained in the sample is still possible. The biomolecules have a very low degree of degradation, thus still RNA isolation (RNA is the molecule which usually has the highest degree of degradation for reason of ubiquitous RNAses) is possible with a very high yield. Comparison with prior compositions, developed for RNA maintenance only, e.g. RNAlater (Ambion), shows that the samples give nearly the same RNA yield, however, different to RNAlater the morphology of the sample is perfectly maintained when the present compositions and method is used. Thus, the same samples can be used for molecular biological as well as for histological analysis. In addition present compositions and methods enable extraction of biomolecules from tiny samples or single cells even after histological analysis e.g. with the use of a laser microdissection device” (see paragraph [0082]), Cory et al., have successfully shown that brain tissue fixed in Bouin’s solution for 5 hr are embedded in paraffin, and 8 µm sections are transferred to silane-coated microscope slides and stained with hematoxylin and eosin (see pages 6 and 7, paragraph [0098]), Chu has successfully shown that “[D]eparaffinized tissue section slides which are in slide holders are placed vertically into a staining dish with 500 mL of hematoxylin solution for 10 seconds with ultrasound followed by washing with running tap water in a staining dish for 5 seconds with ultrasound. The slides are placed in 95% ethyl alcohol for 5 seconds with ultrasound and counterstained in eosin-phloxine solution for 10 seconds with ultrasound” (see column 18), Espina et al., teach that preservation of RNA in a tissue is evaluated by calculating the RNA integrity number (RIN) of a RNA and the RIN of the RNA fixed by their method is compared with the RIN of the RNA preserved by a commercially available, room temperature RNA preservation method, “RNALater®”, and the RIN of an untreated RNA (see paragraphs [0014], [0044] and [0045] and Figure 10), Kennedy et al., teach that RNA quality can be measured by a calculated RIN and “[A]dvantages of RIN include obtain a numerical assessment of the integrity of RNA; directly comparing RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs; and ensuring repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used” (see paragraph [0111]), and the simple substitution of one kind of fixing method (ie., the fixing method using DSP taught by Xiang et al.,) from another kind of fixing method (ie., the fixing method using the non-crosslinking composition comprising methanol and then contacted the biological sample with an ethanol-containing composition taught by Lenz et al.,) during the process of isolating biomolecules from target cells or tissue of a biological sample recited in claim 15, in the absence of convincing evidence to the contrary, would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made since the fixing method using DSP taught by Xiang et al., and the fixing method using the non-crosslinking composition comprising methanol and then contacted the biological sample with an ethanol-containing composition taught by Lenz et al., are used for the same purpose (ie., fixing a biological sample) and are exchangeable. One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to fix the biological sample taught by Xiang et al., using a non-crosslinking composition comprising methanol, then contacted the biological sample with an ethanol-containing composition, and embed the biological sample in paraffin mentioned in the methods taught by Lenz et al., mount the paraffin embedded sample on a support, stain said section recited in claim 15 for a time period that is less than 15 seconds with a first tissue staining agent (ie., hematoxylin) and rinse said section with water, and stain said section recited in claim 15 for a time period that is less than 1 seconds with a second tissue staining agent (ie., Eosin) based on his or her experimental requirement, and comparing an RIN value of the RNA isolated from target cells or tissues in a biological sample using the method recited in claim 15 with an RIN value of the RNA isolated from target cells or tissues in a biological sample using a method different from the method recited in claim 15 in view of the prior arts of Xiang et al., Lenz et al., Cory et al., Chu, Espina et al., and Kennedy et al., so that the methods recited in claims 15, 19, 38, and 44-46 would be performed in order to perfectly maintain the morphology of the biological sample, reduce the degradation of isolated biomolecules comprising RNA, increase yield of the isolated biomolecules comprising RNA and check quality of the isolated RNA such that the target cells or tissue comprising biomolecules of interest would be easily determined by observing a better image of the at least one part of the biological sample mounted on the at least one support stained and rinsed in step (3) of claim 15.
Furthermore, the motivation to make the substitution cited above arises from the expectation that the prior art elements will perform their expected functions to achieve their expected results when combined for their common known purpose. Support for making the obviousness rejection comes from the M.P.E.P. at 2144.06, 2144.07 and 2144.09.
Also note that there is no invention involved in combining old elements is such a manner that these elements perform in combination the same function as set forth in the prior art without giving unobvious or unexpected results. In re Rose 220 F.2d. 459, 105 USPQ 237 (CCPA 1955).
More particularly, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Where the general conditions of a claim are disclosed in the prior art, it is not inventive, in the absence of an unexpected result, to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Claims 15, 17, 19, 20, 23-28, 38, 43-45, 47, and 48 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Xiang et al., in view of Lenz et al., Cory et al., Abate-Shen et al., (US 2008/0216180 A1, published on September 4, 2006), Espina et al., and Kennedy et al..
Regarding claims 15, 17, 20, 23-28, and 43, Xiang et al., teach (1) mounting at least one part of the biological sample (ie., supraoptic nucleus (SON) from brain) on at least one support (ie., the glass slide), (2) optionally treating the at least one part of the biological sample with a sample treatment agent (eg., ethanol or xylene), wherein said treatment can be carried out at any stage of the procedure before step (6), (3) staining the at least one part of the fixed biological sample mounted on the at least one support (ie., stained with anti-oxytocin-neurophysin antibody), (4) determining target cells or tissue comprising biomolecules of interest by observing the at least one part stained in step (3) (ie., red color stained by anti-oxytocin-neurophysin antibody), (5) separating the target cells or tissue (ie., the tissue stained by anti-oxytocin-neurophysin antibody) comprising biomolecules of interest (ie., RNA) from the remainder of the biological sample mounted on the at least one support, and (6) isolating the biomolecules from the target cells or tissue separated in step (5) wherein the biomolecules comprise RNA as recited in claim 15, the method comprises step (2) and the sample treatment agent is a de-waxing agent (ie., xylene) as recited in claim 17 wherein the de-waxing agent is a de-paraffinizing agent such as xylene as recited in claims 20 and 23 (see page 33, paragraph [0375]), the at least one support (ie., the glass slide) is transparent as recited in claim 24, the at least one support is made of glass (ie., the glass slide) or a transparent polymer as recited in claim 25, the target cells or tissue comprising biomolecules of interest from the biological sample in step (4) is observed under light or backlight (eg., by using Leica DM RX microscope) as recited in claim 26, the target cells or tissue comprising biomolecules of interest from the biological sample in step (4) is observed under a microscope (eg., Leica DM RX microscope) as recited in claim 27, the target cells or tissue is/are separated in step (5) by microdissection, laser capture microdissection, scraping, or cutting out as recited in claim 28, and the biological sample comprises at least one section of 0.5 to 12 µm (ie., 8 µm) thickness of a non-formalin-fixed paraffin-embedded sample as recited in claim 43 (see pages 32-36, Example 14, paragraphs [0368] to [390]).
Regarding claim 38, Xiang et al., teach that the fixed biological sample is divided in sections of 0.5 to 12 µm thickness (ie., 8µm), and in step (1), the at least one of the support is a transparent support, in step (2), the section is treated with the sample treatment agent and the sample treatment agent is a de-waxing agent (ie., xylene), and in step (4), the target cells or tissue is/are determined in the stained sample section (see pages 32-36, Example 14, paragraphs [0368] to [390]). Although Xiang et al., do not teach that, in step (5) of claim 15, the remainder of the fixed sample (ie., fixed sections stained by anti-oxytocin-neurophysin antibody) is discarded, in view of the teachings of Xiang et al., it is obvious to one having ordinary skill in the art at the time the invention was made to discard the slides after scraping fixed sections stained by anti-oxytocin-neurophysin antibody from the slides into Eppendorf tubes (see paragraph [0371]).
Xiang et al., do not disclose that the biological sample is fixed with a non-crosslinking composition with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition, and a wax embedded and staining the at least one part of the biological sample mounted on the at least one support for not more than 60 seconds with at least one cell or tissue staining agent and rinsing the at least one part of the biological sample with water wherein the non-crosslinking composition does not comprise formaldehyde, the isolated RNA has an RNA Integrity Number (RIN) value comparable to a reference RIN value, and the reference RIN value is the RIN value of RNA that is isolated from the biological sample as recited in claim 15, the fixed biological sample is non-formalin-fixed paraffin-embedded sample (non-FFPE- sample) as recited in claim 19, step (3), said section is stained for not more than 60 seconds with a first tissue staining agent and rinsed said section with water as recited in claim 38, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for less than 45 seconds with the at least one cell or tissue staining agent as recited in claim 44, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for less than 30 seconds with the at least one cell or tissue staining agent as recited in claim 45, step (3) comprises: (a) staining the at least one part of the biological sample mounted on the at least one support for up to 30 seconds with Hematoxylin, (b) rinsing the at least one part of the biological sample stained in step (a) with water, and (c) staining the at least one part of the biological sample rinsed in step (b) for less than 3 seconds with Eosin as recited in claim 47, and step (c) comprises staining the at least one part of the biological sample rinsed in step (b) for less than 1 second with Eosin as recited in claim 48.
Lenz et al., teach that a biological sample is fixed with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition, and wax embedded (see abstract, Examples 8 and 9, and claims 1-21).
Regarding claims 15 and 19, since Cory et al., teach that brain tissue fixed in Bouin’s solution for 5 hr are embedded in paraffin, and 8 µm sections are transferred to silane-coated microscope slides and stained with hematoxylin and eosin (see pages 6 and 7, paragraph [0098]), Cory et al., disclose that the fixed biological sample is a wax embedded sample as recited in claim 15 and the fixed biological sample is non-formalin-fixed paraffin-embedded sample (non-FFPE- sample) as recited in claim 19.
Regarding claims 15, 38, 44, 45, and 47, since Abate-Shen et al., teach that “[F]rozen sections were allowed to thaw briefly for 1 hour. Slides were then rehydrated (100% ethanol 2×for 2 minutes, 95% EtOH for 2 minutes, 70% ethanol for 2 minutes, 50% ethanol for 2 minutes, Milli-Q diH2O 2 ×for 5 minutes). Staining was as follows: slides immersed in filtered Harris Modified Hematoxylin (Fisher SH26D-500) for ̴ 15 seconds, rinsed with Milli-Q diH2O until water, immersed in Eosin for 5 seconds, dehydrated in ascending alcohol solutions
(95%, 2×100% for 2 mins each), cleared with xylene 3×for 5 minutes each, and coverslipped with Clear Mount” (see paragraph [0181]), Abate-Shen et al., disclose staining the at least one part of the biological sample mounted on the at least one support for not more than 60 seconds with at least one cell or tissue staining agent and rinsing the at least one part of the biological sample with water as recited in claim 15, staining the at least one part of the biological sample mounted on the at least one support for not more than 60 seconds with at least one cell or tissue staining agent and rinsing the at least one part of the biological sample with water as recited in claim 15 wherein said section is stained for not more than 60 seconds with a first tissue staining agent and rinsed said section with water as recited in claim 38, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for less than 45 seconds with the at least one cell or tissue staining agent as recited in claim 44, step (3) comprises staining the at least one part of the biological sample mounted on the at least one support for less than 30 seconds with the at least one cell or tissue staining agent as recited in claim 45, step (3) comprises: (a) staining the at least one part of the biological sample mounted on the at least one support for up to 30 seconds with Hematoxylin, and (b) rinsing the at least one part of the biological sample stained in step (a) with water as recited in claim 47.
Espina et al., teach that preservation of RNA in a tissue is evaluated by calculating the RNA integrity number (RIN) of a RNA and the RIN of the RNA fixed by their method is compared with the RIN of the RNA preserved by a commercially available, room temperature RNA preservation method, “RNALater®”, and the RIN of an untreated RNA (see paragraphs [0014], [0044] and [0045] and Figure 10).
Kennedy et al., teach that RNA quality can be measured by a calculated RIN and “[A]dvantages of RIN include obtain a numerical assessment of the integrity of RNA; directly comparing RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs; and ensuring repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used” (see paragraph [0111]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the methods as recited in claims 15, 19, 38, 44, 45, 47, and 48 by staining the at least one part of the biological sample mounted on the at least one support for less than 30 seconds with at least one cell or tissue staining agent or hematoxylin, rinsing the at least one support with water, and staining the at least one part of the biological sample rinsed for less than 1 second with Eosin wherein the biological sample is fixed with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition and a wax embedded, the non-crosslinking composition does not comprise formaldehyde, and is not Methacarn solution, and the fixed biological sample is non-formalin-fixed paraffin-embedded sample (non-FFPE-sample such as DSP-fixed paraffin-embedded biological sample), the isolated RNA has an RNA Integrity Number (RIN) value comparable to a reference RIN value, and the reference RIN value is the RIN value of RNA that is isolated from the biological sample in view of the prior arts of Xiang et al., Lenz et al., Cory et al., Abate-Shen et al., Espina et al., and Kennedy et al.. One having ordinary skill in the art would have been motivated to do so because Lenz et al., have successfully fixed a biological sample with a non-crosslinking composition comprising methanol, then contacted with an ethanol-containing composition, and wax embedded (see abstract, Examples 8 and 9, and claims 1-21), and provided advantages of their composition and the method “[T]he present compositions and method provide samples which are treated in a way that isolation of most of the biological components originally contained in the sample is still possible. The biomolecules have a very low degree of degradation, thus still RNA isolation (RNA is the molecule which usually has the highest degree of degradation for reason of ubiquitous RNAses) is possible with a very high yield. Comparison with prior compositions, developed for RNA maintenance only, e.g. RNAlater (Ambion), shows that the samples give nearly the same RNA yield, however, different to RNAlater the morphology of the sample is perfectly maintained when the present compositions and method is used. Thus, the same samples can be used for molecular biological as well as for histological analysis. In addition present compositions and methods enable extraction of biomolecules from tiny samples or single cells even after histological analysis e.g. with the use of a laser microdissection device” (see paragraph [0082]), Cory et al., have successfully shown that brain tissue fixed in Bouin’s solution for 5 hr are embedded in paraffin, and 8 µm sections are transferred to silane-coated microscope slides and stained with hematoxylin and eosin (see pages 6 and 7, paragraph [0098]), Abate-Shen et al., successfully shown that “[F]rozen sections were allowed to thaw briefly for 1 hour. Slides were then rehydrated (100% ethanol 2×for 2 minutes, 95% EtOH for 2 minutes, 70% ethanol for 2 minutes, 50% ethanol for 2 minutes, Milli-Q diH2O 2 ×for 5 minutes). Staining was as follows: slides immersed in filtered Harris Modified Hematoxylin (Fisher SH26D-500) for ̴ 15 seconds, rinsed with Milli-Q diH2O until water, immersed in Eosin for 5 seconds, dehydrated in ascending alcohol solutions (95%, 2×100% for 2 mins each), cleared with xylene 3×for 5 minutes each, and coverslipped with Clear Mount” (see paragraph [0181]), Espina et al., teach that preservation of RNA in a tissue is evaluated by calculating the RNA integrity number (RIN) of a RNA and the RIN of the RNA fixed by their method is compared with the RIN of the RNA preserved by a commercially available, room temperature RNA preservation method, “RNALater®”, and the RIN of an untreated RNA (see paragraphs [0014], [0044] and [0045] and Figure 10), Kennedy et al., teach that RNA quality can be measured by a calculated RIN and “[A]dvantages of RIN include obtain a numerical assessment of the integrity of RNA; directly comparing RNA samples, e.g. before and after archival, compare integrity of same tissue across different labs; and ensuring repeatability of experiments, e.g. if RIN shows a given value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used” (see paragraph [0111]), and the simple substitution of one kind of fixing method (ie., the fixing method using DSP taught by Xiang et al.,) from another kind of fixing method (ie., the fixing method using the non-crosslinking composition comprising methanol and then contacted the biological sample with an ethanol-containing composition taught by Lenz et al.,) during the process of isolating biomolecules from target cells or tissue of a biological sample recited in claim 15, in the absence of convincing evidence to the contrary, would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made since the fixing method using DSP taught by Xiang et al., and the fixing method using the non-crosslinking composition comprising methanol and then contacted the biological sample with an ethanol-containing composition taught by Lenz et al., are used for the same purpose (ie., fixing a biological sample) and are exchangeable. One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to fix the biological sample taught by Xiang et al., using the non-crosslinking composition comprising methanol, then contacted the biological sample with an ethanol-containing composition, and embed the biological sample in paraffin and embed the biological sample fixed by the non-crosslinking fixative composition in paraffin mentioned in the methods taught by Lenz et al., mount the paraffin embedded sample on a support, stain the at least one part of the biological sample mounted on the at least one support for a time period that is less than 15 seconds with a first cell or tissue staining agent and rinse the at least one support with water, and stain said section recited in claim 15 for a time period that is less than 1 seconds with a second tissue staining agent (ie., Eosin) based on his or her experimental requirement and comparing an RIN value of the RNA isolated from target cells or tissues in a biological sample using the method recited in claim 15 with an RIN value of the RNA isolated from target cells or tissues in a biological sample using a method different from the method recited in claim 15 in view of the prior arts of Xiang et al., Lenz et al., Cory et al., Abate-Shen et al., Espina et al., and Kennedy et al., so that the methods recited in claims 15, 18, 38, 44, 45, 47, and 48 would be performed in order to perfectly maintain the morphology of the biological sample, reduce the degradation of isolated biomolecules comprising RNA, increase yield of the isolated biomolecules comprising RNA, and check quality of the isolated RNA such that the target cells or tissue comprising biomolecules of interest would be easily determined by observing a better image of the at least one part of the biological sample mounted on the at least one support stained and rinsed in step (3) of claim 15.
Furthermore, the motivation to make the substitution cited above arises from the expectation that the prior art elements will perform their expected functions to achieve their expected results when combined for their common known purpose. Support for making the obviousness rejection comes from the M.P.E.P. at 2144.06, 2144.07 and 2144.09.
Also note that there is no invention involved in combining old elements is such a manner that these elements perform in combination the same function as set forth in the prior art without giving unobvious or unexpected results. In re Rose 220 F.2d. 459, 105 USPQ 237 (CCPA 1955).
More particularly, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Where the general conditions of a claim are disclosed in the prior art, it is not inventive, in the absence of an unexpected result, to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)
Response to Arguments
I. In page 9, seventh paragraph bridging to page 11, first paragraph of applicant’s remarks, applicant argues that “one of ordinary skill in the art would not have been motivated to substitute the fixative DSP of Xiang with that of Lenz because doing so would change the primary teaching of Xiang and thus eliminate the associated advantages. Briefly, Xiang relates to labeling nucleic acid molecules for use in hybridization reactions and is not closely related to the present application. Example 14 of Xiang on pages 32- 36 relates to using Dithio-bis(Succinimidyl Propionate) (DSP, a crosslinking fixative) to fix tissue section for immunostaining, microdissection and expression profiling. This Example was concerned with fixing tissue sections for immunostaining, such as sections intended for laser capture microdissection (LCM), to extract intact, unmodified RNA (see paragraph [0368]). Immunostaining involves recognizing cell populations of interest within the tissue sections using dye conjugated antibodies (see paragraphs [0370], [0375], [0381], and [0382]). Example 14 shows that (1) ethanol fixation was not satisfactory in immunostaining the tissue, and also resulted in degradation of RNA isolated from the fixed sample (see paragraphs [0381], [0382] and [0384]), (2) it was difficult, if not impossible, to extract RNA from formalin (i.e., formaldehyde) fixed tissues (see paragraphs [0384] and [0369]), but (3) DSP fixed soluble antigens and protected RNA in tissue sections, even ones that have been immunostained (see paragraph [0390]). Xiang describes their DSP fixing method as ‘a novel, fast, and simple method to fix and immunostain tissue sections, such as sections intended for laser capture microdissection, in order to extract intact, unmodified RNA’ (see paragraph [0368], emphasis added). Thus, Xiang shows that ethanol as a non-crosslinking fixing reagent is not suitable, one crosslinking fixing reagent (formalin) is not suitable either, but another crosslinking fixing reagent (DSP) is suitable, for isolating RNA from a biological material fixed with the indicated fixing reagent and then immunostained. Therefore, Xiang teaches that neither crosslinking nor non-crosslinking fixatives can be easily exchanged when intending to isolate RNA from a correspondingly fixed and immunostained sample. In view of the above-noted lack of interchangeability among fixatives disclosed in Xiang, one skilled in the art would not have been motivated to use another fixative (e.g., the non-crosslinking fixative and treatment as disclosed in Lenz) in the method of Xiang without experimental evidence showing that the other fixative is suitable for fixing biological samples for immunostaining followed by isolating RNA from immunostained tissue. Doing so would change the primary teaching of using DSP in Xiang and thus eliminate the associated advantages. In addition, one skilled in the art would not have been motivated to use the fixative and related treatment of Lenz in the method of Xiang because Xiang teaches away using methanol-or ethanol-based fixatives. Xiang discloses that ‘fixing cells or tissue sections with precipitating fixatives (for example, alcohol, acetone, etc.) results in the diffusion of small, soluble molecules including antigens, such as peptides’ (see paragraph [0363]) (emphasis added). Xiang reiterates its purpose ‘to generate a protocol, whereby the cell can be fixed in such a way that RNA can be preserved (for subsequent harvest and analysis) without the loss of cell morphology or other cell contents’ (Id., emphasis added). Because methanol is a precipitating agent,1 Xiang teaches away from using methanol as a fixative. In addition, Example 13 of Xiang shows that ethanol was unsatisfactory in fixing tissue for immunostaining (see e.g., paragraph [0366], indicating soluble antigens could not be stained in ethanol-fixed cells). In view of the failure in immunostaining and identifying target cells using an alcohol- containing fixative as disclosed in Xiang, one of ordinary skill in the art would not have expected to isolate RNA from such target cells. As such, such a person would not have been motivated to replace the fixative DSP of Xiang with an alcohol-containing fixative of Lenz”.
These arguments have been fully considered but they are not persuasive toward the
withdrawal of the rejections.
First, although applicant argues that “ethanol fixation was not satisfactory in immunostaining the tissue, and also resulted in degradation of RNA isolated from the fixed sample (see paragraphs [0381], [0382] and [0384])” and “[E]xample 13 of Xiang shows that ethanol was unsatisfactory in fixing tissue for immunostaining (see e.g., paragraph [0366], indicating soluble antigens could not be stained in ethanol-fixed cells)”, since Xiang et al., teach that “[T]he morphology of DSP-, ethanol-, and formalin-fixed cells was well preserved. Immunostaining with an anti-beta-actin antibody indicated that soluble antigens could be stained in DSP-fixed but no in ethanol-fixed cells’ and “[E]thanol fixation was even less satisfactory when an antibody directed against the oxytocin-neurophysin was used to stain hypothalamic magnocellular neurons in sections prepared from frozen rat brains” (see paragraphs [0366] and [0382]), Xiang et al., clearly indicate that ethanol fixation is not satisfactory in immunostaining the tissue only when anti-beta-actin antibody or an antibody directed against the oxytocin-neurophysin is used. In fact, claim 15 does not require that the staining step is performed using an antibody while claim 47 requires that the first cell or tissue staining agent is Hematoxylin and the second cell or tissue staining agent is Eosin.
Second, although applicant argues that “[X]iang discloses that ‘fixing cells or tissue sections with precipitating fixatives (for example, alcohol, acetone, etc.) results in the diffusion of small, soluble molecules including antigens, such as peptides’ (see paragraph [0363]) (emphasis added). Xiang reiterates its purpose ‘to generate a protocol, whereby the cell can be fixed in such a way that RNA can be preserved (for subsequent harvest and analysis) without the loss of cell morphology or other cell contents’ (Id., emphasis added). Because methanol is a precipitating agent,1 Xiang teaches away from using methanol as a fixative”, paragraph [0363] of Xiang et al., does not indicate that precipitating fixatives (e.g., alcohol) results in the loss of cell morphology or other cell contents such that RNA cannot be preserved and results in the diffusion. In fact, RNA has been successfully isolated from the biological sample fixed using the method taught by Lenz et al., (using the non-crosslinking composition comprising methanol and then contacted the biological sample with an ethanol-containing composition) (see page 12).
II. In page 11, second paragraph bridging to page 13, first paragraph of applicant’s remarks, applicant argues that “one of ordinary skill in the art would not have had a reasonable expectation of success to modify Xiang in view of Lenz to arrive at the presently claimed methods. As indicated above, the claimed method allows for isolating RNA from a biological
sample after being stained with an RNA Integrity Number (RIN) value comparable to the
reference RIN value of RNA isolated directly from the biological sample. Applicant submits
that one of ordinary skill in the art would not have a reasonable expectation to modify Xiang in
view of Lenz to arrive at the methods currently claimed in the present application. Specifically, RNA degradation is the one of the major issues when fixing and staining a biological sample to prepare for laser capture microdissection (LCM). See Kihara et al., Journal of Neuroscience Methods 148, 103-107 (enclosed in the response filed December 3, 2024). In Kihara, tissue samples were fixed in 2% paraformaldehyde (a crosslinking agent) to prevent RNA degradation during the fixation and subsequent processing (see Abstract). In some cases, even when a tissue is stabilized using formalin (another crosslinking agent), degradation of RNA still occurred when isolating RNA from the stabilized tissue as shown in Example 14 of Xiang. Thus, formalin-fixed tissues are typically used for histological analyses rather than for isolating biomolecules. In particular, formalin-fixed tissues are usually not suitable for isolating non-degraded RNA.
As one skilled in the art appreciated, it is critical to ensure RNase inhibition and using RNase free surroundings during staining. See e.g., Applied Biosystems, From postmortem homogenous cells to microarray analysis: an optimized protocol, 6 pages, 2015, enclosed in the response filed December 3, 2024. This shows how sensitive the RNA is to the conditions of the staining procedure. When using a non-formalin fixed tissue, such as those treated with the compositions disclosed in Lenz, the tissue can be stabilized without RNA being degraded. Thus, the non- formalin fixed paraffin embedded tissue may be used in histological analyses as well as in isolating biomolecules including RNA. Because a fixation with non-crosslinking fixatives is not long-term but reversible, a skilled person would expect that once the fixing conditions are no longer maintained, particularly in aqueous surroundings like in the staining medium, RNase would be re-activated and degrade RNA. Therefore, one should directly start isolating the RNA after removing the fixed sample material from the fixation medium under conditions that block the RNase activity, such as the use of Trizol as cited in Xiang (see paragraph [0370]) (Trizol contains a chaotropic agent and, consequently, inhibits RNase activity). Alternatively, such a person may embed the fixed tissue, such as in wax like paraffin under water-free conditions for long term storage. However, once the wax is removed for further analysis, it is important to ensure that RNA is isolated under conditions in which the RNase is not re-activated. The present inventors surprisingly found that the methods currently claimed in the present application allows isolating RNA from a biological sample after being stained with an RIN value comparable to that without being stained. For example, Example 2 shows RNA isolation and histomorphology analysis from sections of tissue after being stained with hematoxylin (for 60 or fewer seconds) and eosin. The results indicate that the tissue sections were successfully stained (see Figure 7), and RNA in the stained sections was preserved and isolated with quality comparable to that from reference sections without staining (see Figure 5 where RIN of RNA isolated from the reference sections without being stained was about 3.7, and RINs of RNA isolated from sections subject to two staining treatments were about 3.8 and about 4.3). Thus, in view of the state of art as of the effective filing date of the present application, especially in view of the belief that RNase would be re-activated after a sample fixed by a non- crosslinking fixative is stained in an aqueous dye solution, one of ordinary skill in the art would not have had a reasonable expectation of success to modify Xiang in view of Lenz (and the other cited references discussed below) to arrive at the presently claimed methods”.
These arguments have been fully considered but they are not persuasive toward the withdrawal of the rejections.
First, since Lenz et al., teach that “[T]his example shows once more the effect of tissue treatment on RNA stability according to the invention, i.e. the transfer from a reagent according to composition A to a reagent according to composition B. In samples where transfer took place the RNA yield remained high in a range comparable to the reference with RNAlater (table 5, 1-7 and 9). No visible RNA degradation could be observed even after 3 days of storage (FIGS. 5, 1-7 and 9)” (see page 12 and Table 5), their method discloses that the biological sample is fixed with a non-crosslinking composition comprising methanol and then contacted with an ethanol-containing composition as recited in claim 15 and can keep RNA stability and prevent RNA degradation after the RNA is isolated and applicant’s argument “one of ordinary skill in the art would not have a reasonable expectation to modify Xiang in view of Lenz to arrive at a method that allows RNA isolated from a biological sample after being stained to have RNA integrity comparable to that of RNA isolated from the biological sample without being stained” is incorrect.
Second, although applicant argues that “in view of the state of art as of the effective filing date of the present application, especially in view of the belief that RNase would be re-activated after a sample fixed by a non- crosslinking fixative is stained in an aqueous dye solution, one of ordinary skill in the art would not have had a reasonable expectation of success to modify Xiang in view of Lenz (and the other cited references discussed below) to arrive at the presently claimed methods”, since the rejection is based on the simple substitution of one kind of fixing method (ie., the fixing method using DSP taught by Xiang et al.,) from another kind of fixing method (ie., the fixing method using the non-crosslinking composition comprising methanol and then contacted the biological sample with an ethanol-containing composition taught by Lenz et al.,) during the process of isolating biomolecules from target cells or tissue of a biological sample recited in claim 15 and is not dependent on replacing one kind of RNA isolation method from another kind of RNA isolation method, a chemical material that blocks the RNase activity such as Trizol as cited in Xiang et al., is still used in step (6) of claim 15 such that the RNase is not re-activated during the process of RNA isolation and one of ordinary skill in the art would have had a reasonable expectation of success to modify Xiang et al., in view of Lenz et al., to arrive at the presently claimed methods.
III. In page 13, second paragraph bridging to page 14, second paragraph of applicant’s remarks, applicant argues that “[T]he deficiencies of lacking both a motivation to modify Xiang in view of Lenz and a reasonable expectation of success to arrive at the presently claimed methods have not been remedied by the other cited references. Specifically, Cory relates to a method and an animal model for identifying molecules and genetic sequences for treating male infertility. The relevant part of Cory is Example 4, which discloses fixing tissue in Bouin’s solution for 5 hours in paraffin, transferring sections of fixed tissue to microscope slides, and staining the tissue sections with Hematoxylin and Eosin. Bouin’s solution comprises formaldehyde, and is thus not a non- crosslinking composition. Thus, Cory is silent with respect to using a non-crosslinking composition in fixing biological samples. Chu does not show that one may successfully extract RNA from a tissue fixed by a non- crosslinking fixative, either, let alone from tissue sections that have been stained and rinsed with water. Chu relates to methods of fixing and processing tissue and samples on a membrane by using ultrasound radiation. This reference is cited in the Office Action as disclosing staining tissue section slides with hematoxylin and eosin in Example 18. Yet, the fixative used in that Example was formalin, which is a crosslinking agent. Abate-Shen relates to a transgenic animal having a somatic cell in which at least one allele of an endogenous p53 and Pten gene is functionally disrupted. Similar to Chu, this reference is also cited as disclosing an H&E staining protocol in paragraph [0181]. However, the tissue sections were either fixed with a crosslinking fixative (i.e., formalin or paraformaldehyde) or snap-frozen (see paragraphs [0180] and [0181]). Espina relates to one-step cell and tissue preservative compositions for morphologic and molecular analysis. The one-step cell and tissue preservative compositions comprise a precipitating fixative and a reversible/ cleavable cross-linker as well as other optional components, such as a permeation enhancer, a kinase inhibitor, and a phosphatase inhibitor. This reference is cited in the Office Action as disclosing evaluating preservation of RNA in a tissue by calculating the RIN and comparing it to an RIN of a control RNA. It does not teach or suggest a motivation to modify Xiang in view of Lenz or provide a reasonable expectation of success to arrive at the presently claimed methods. Kennedy relates to methods and compositions of molecular profiling for disease diagnostics. It is cited in the Office Action as teaching that RNA quality can be measured by a calculated RIN. It fails to teach or suggest a motivation to modify Xiang in view of Lenz or provide a reasonable expectation of success to arrive at the presently claimed methods”.
These arguments have been fully considered but they are not persuasive toward the withdrawal of the rejections because references from Cory et al., Chu, Abate-Shen et al., Espina et al., and Kennedy et al., are not related to the simple substitution of one kind of fixing method from another kind of fixing method in the rejection but are used to reject other parts of claim 15 which are not taught by Xiang et al., and Lenz et al..
IV. In page 14, fourth paragraph bridging to page 16, first paragraph of applicant’s remarks, applicant argues that “[A]pplicant submits that one skilled in the art would not interpret Xiang narrowly as teaching that ‘ethanol fixation is not satisfactory in immunostaining the tissue only when anti-beta-actin antibody or an antibody directed against the oxytocin-neurophysin is used’ as asserted in the Office Action. Xiang discloses that ‘fixing cells or tissue sections with precipitating fixatives (for example, alcohol, acetone, etc.) results in the diffusion of small, soluble molecules including antigens, such as peptides’ (see paragraph [0363]) (emphasis added). Xiang further states that its purpose is ‘to generate a protocol, whereby the cell can be fixed in such a way that RNA can be preserved (for subsequent harvest and analysis) without the
loss of cell morphology or other cell contents’ (Id., emphasis added). In addition, Example 13 of Xiang shows that ethanol was unsatisfactory in fixing tissue for immunostaining and states ‘soluble antigens could be stained in DSP-fixed but no in ethanol-fixed cells’ (see e.g., paragraph [0366], emphasis added). In view of the above-noted general description in Xiang (e.g., using the generic terms of ‘other cell contents’ and ‘soluble antigens,’ one of ordinary skill in the art would read Xiang as teaching or suggesting that alcohol-containing fixatives are not suitable for immunostaining soluble antigens (e.g., peptides) in biological samples in general as opposed to limiting the antigens to only actin and oxytocin-neurophysin as asserted in the Office Action. In addition, Applicant submits that the fact that claim 15 does not require that the staining step is performed using an antibody is not material to obviousness determination in this case. The question at issue is whether one of ordinary skill in the art would have been motivated to modify Xing in view of Lenz to arrive at the presently claimed methods. As discussed above, Applicant submits that no sufficient motivation is provided for establishing a prima facie case of obviousness against the instant claims because doing so would change the primary teaching of Xiang and thus eliminate the associated advantages. Applicant disagrees with the assertion in the Office Action that ‘Lenz et al., have clearly shown that ethanol fixation is satisfactory in immunostaining the tissue when this tissue has been stained with Hematoxyli and Eosin (see paragraph [0155] and Figure 11).’ Paragraph [0155] and Figure 11 of Lenz do not disclose ethanol fixation followed by immunostaining the tissue and thus cannot show that ethanol fixation is satisfactory in immunostaining the tissue. Hemotoxylin and eosin stain is a tissue stain that combines two histological stains: hematoxylin and eosin (see H&E stain, Wikipedia,
en.wikipedia.org/wiki/H%26E_stain, enclosed in Applicant's response filed February 26, 2024).
The hematoxylin stains cell nuclei (e.g., DNA) a purplish blue, and eosin stains the extracellular matrix (e.g., collagen) and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors. Id. Immunostaining, on the other hand, refers to the process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, then visualizing the bound antibody using a chromogen (immunohistochemistr ) or fluorescence (immunofluorescence) (see Idleburg et al., Methods Mol Biol. 2221:261-73, 2021, enclosed, Abstract). Thus, staining tissue with hematoxylin and eosin as described in paragraph [0155] of Lenz is not immunostaining”.
These arguments have been fully considered but they are not persuasive toward the withdrawal of the rejections.
First, although applicant argues that “one skilled in the art would not interpret Xiang narrowly as teaching that ‘ethanol fixation is not satisfactory in immunostaining the tissue only when anti-beta-actin antibody or an antibody directed against the oxytocin-neurophysin is used’ as asserted in the Office Action” and “[I]n view of the above-noted general description in Xiang (e.g., using the generic terms of ‘other cell contents’ and ‘soluble antigens,’ one of ordinary skill in the art would read Xiang as teaching or suggesting that alcohol-containing fixatives are not suitable for immunostaining soluble antigens (e.g., peptides) in biological samples in general as opposed to limiting the antigens to only actin and oxytocin-neurophysin as asserted in the Office Action”, paragraphs [0363] and [0382] of Xiang et al., only teaches that ethanol fixation is not satisfactory in immunostaining the tissue when anti-beta-actin antibody or an antibody directed against the oxytocin-neurophysin is used. In fact, claim 15 does not require that the staining step is performed using an antibody while claim 47 requires that the first cell or tissue staining agent is Hematoxylin and the second cell or tissue staining agent is Eosin.
Second, although applicant argues that “[P]aragraph [0155] and Figure 11 of Lenz do not disclose ethanol fixation followed by immunostaining the tissue and thus cannot show that ethanol fixation is satisfactory in immunostaining the tissue. Hemotoxylin and eosin stain is a tissue stain that combines two histological stains: hematoxylin and eosin” and “[I]mmunostaining, on the other hand, refers to the process of identifying proteins in tissue sections by incubating the sample with antibodies specific to the protein of interest, then visualizing the bound antibody using a chromogen (immunohistochemistr ) or fluorescence (immunofluorescence) (see Idleburg et al., Methods Mol Biol. 2221:261-73, 2021, enclosed, Abstract). Thus, staining tissue with hematoxylin and eosin as described in paragraph [0155] of Lenz is not immunostaining”, claim 15 does not require that the staining step is performed by immunostaining the tissue as argued by applicant. In fact, dependent claim 47 requires that the first cell or tissue staining agent is Hematoxylin and the second cell or tissue staining agent is Eosin. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
V. In page 16, second paragraph bridging to page 18, second paragraph of applicant’s remarks, applicant argues that “[P]aragraph [0363] of Xiang indicates that its goal is to generate a protocol whereby the cell can be fixed in such a way that RNA can be preserved (for subsequent harvest and analysis) without the loss of cell morphology or other cell contents. One of ordinary skill in the art would not be motivated to use alcohol in fixing cells in view of the additional teaching in Xiang that fixing cells or tissue sections with precipitating fixatives such as alcohol results in the diffusion of small, soluble molecules including antigens, such as peptides. In addition, Xiang compared three fixatives and found that DSP but not ethanol fixation enabled RNA isolation after immunostaining (see Example 14). In view of the teachings in Xiang, contrary to the assertion in the Office Action, one skilled in the art would read paragraph [0363] of Xiang as teaching precipitating fixatives (e.g., alcohol) results in the loss of cell morphology or other cell contents such that RNA cannot be extracted from fixed cellular material that has not lost its cell morphology or other cell contents” and “[F]irst, Applicant respectfully disagrees with the contention in the Office Action that because Lenz discloses isolating RNA from a biological sample fixed with a non- crosslinking composition, one of ordinary skill in the art would have a reasonable expectation of modifying Xiang in view of Lenz to arrive at the instantly claimed methods. As discussed above, one skilled in the art would be concerned with reactivation of RNase during the staining step, which is not addressed in Lenz.
Second, Applicant respectfully disagrees with the contention in the Office Action that because the rejection is based on the simple substitution of one kind of fixing method (i.e., DSP taught by Xiang with the fixative of Lenz) and is not dependent on replacing one kind of RNA isolation method for another kind of RNA isolation method, a chemical material that blocks the RNase activity such as Trizol as cited in Xiang is still used in step (6) of claim 15 so that RNase is not re-activated during RNA isolation, one skilled in the art would have had a reasonable expectation of success to modify Xiang in view of Lenz to arrive at the presently claimed methods. As discussed above, one of ordinary skill in the art would not have substituted the fixing method of Xiang with that of Lenz; this is not a case of simple substitution of fixing methods as asserted in the Office Action. In addition, re-activation of RNase was expected to happen during the staining step for identifying the region from which biomolecules like RNA should be isolated. Using a chemical material that blocks the RNase activity such as Trizol as in Xiang in step (6) of instant claim 15 would not address the concern of RNA degradation in the staining step. Instead, using a lysis buffer or Trizol at this step would damage or destroy cellular structures of the fixed sample on the slide, defeating the purpose of performing this step”.
These arguments have been fully considered but they are not persuasive toward the withdrawal of the rejections.
First, although applicant argues that “[I]n view of the above-noted general description in Xiang (e.g., using the generic terms of ‘other cell contents’ and ‘soluble antigens,’ one of ordinary skill in the art would read Xiang as teaching or suggesting that alcohol-containing fixatives are not suitable for immunostaining soluble antigens (e.g., peptides) in biological samples in general as opposed to limiting the antigens to only actin and oxytocin-neurophysin as asserted in the Office Action” and paragraph [0363] of Xiang et al., states that “fixing cells or tissue sections with precipitating fixatives (for example, alcohol, acetone, etc.) results in the diffusion of small, soluble molecules including antigens, such as peptides”, Xiang et al., do not disclose that “precipitating fixatives (e.g., alcohol) results in the loss of cell morphology or other cell contents such that RNA cannot be extracted from fixed cellular material that has not lost its cell morphology or other cell contents” as argued by applicant.
Second, although applicant argues that “[A]s discussed above, one skilled in the art would be concerned with reactivation of RNase during the staining step, which is not addressed in Lenz” and “[A]s discussed above one of ordinary skill in the art would not have substituted the fixing method of Xiang with that of Lenz; this is not a case of simple substitution of fixing methods as asserted in the Office Action. In addition, re-activation of RNase was expected to happen during the staining step for identifying the region from which biomolecules like RNA should be isolated. Using a chemical material that blocks the RNase activity such as Trizol as in Xiang in step (6) of instant claim 15 would not address the concern of RNA degradation in the staining step. Instead, using a lysis buffer or Trizol at this step would damage or destroy cellular structures of the fixed sample on the slide, defeating the purpose of performing this step”, claim 15 does not require that the isolated RNA is RNA which does not have degraded RNA molecules argued by applicant. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Conclusion
All claims are identical to or patentably indistinct from, or have unity of invention with claims in the application prior to the entry of the submission under 37 CFR 1.114 (that is, restriction (including a lack of unity of invention) would not be proper) and all claims could have been finally rejected on the grounds and art of record in the next Office action if they had been entered in the application prior to entry under 37 CFR 1.114. Accordingly, THIS ACTION IS MADE FINAL even though it is a first action after the filing of a request for continued examination and the submission under 37 CFR 1.114. See MPEP § 706.07(b). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
No claim is allowed.
Papers related to this application may be submitted to Group 1600 by facsimile transmission. Papers should be faxed to Group 1600 via the PTO Fax Center. The faxing of such papers must conform with the notices published in the Official Gazette, 1096 OG 30 (November 15, 1988), 1156 OG 61 (November 16, 1993), and 1157 OG 94 (December 28, 1993)(See 37 CAR § 1.6(d)). The CM Fax Center number is (571)273-8300.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Frank Lu, Ph.D., whose telephone number is (571)272-0746. The examiner can normally be reached on Monday-Friday from 9 A.M. to 5 P.M.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Dr. Anne Gussow, Ph.D., can be reached on (571)272-6047.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/FRANK W LU/Primary Examiner, Art Unit 1683 May 29, 2026
Prosecution Insights