The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
The title of the invention is not descriptive. A new title is required that is clearly indicative of the invention to which the claims are directed.
The disclosure is objected to because of the following informalities: in instant paragraph [0120], the first sentence of the paragraph includes the following “the second fluorescent dye is” and “the first fluorescent dye is”. Since instant paragraph [0120] appears to be describing the second fluorescent dye and because at least instant paragraph [0006] teaches that the second fluorescent dye is a fluorescent dye that specifically binds to RNA, “the first fluorescent dye” language in instant paragraph [0120] appears to be incorrect.
Appropriate correction is required.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1-39 are rejected under 35 U.S.C. 112(a) because the specification, while being enabling for a method in which a lysing agent is contacted with the sample, does not reasonably provide enablement for a scope that does not include a lysing agent. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
In order to determine compliance with the enablement requirement of 35 U.S.C. 112(a), the Federal Circuit developed a framework of factors in In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988), referred to as the Wands factors to assess whether any necessary experimentation required by the specification is "reasonable" or is "undue." These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
With respect to the breadth of the claims Wands factor, the instant claims cover a scope in which the sample is not brought into contact with a lysing agent. In other words, for the first and second dye to stain the respective DNA or RNA of the cells, this must be able to occur without lysis of at least the red blood cells in the sample and/or permeation of the membranes of white blood cells. The claims do not define what if any characteristics of the first fluorescence information, the second fluorescence information and the scattered light information are utilized to specify a particle and output indicating presence or absence of a first abnormal cell a second abnormal cell or a third abnormal cell.
With respect to the state of the prior art Wands factor, paragraph [0042] of the cited Kimura patent publication (US 2018/0356328) teaches that by using a described hemolytic agent, red blood cells are hemolyzed, the cell membranes of nucleated cells can be damaged, and the nucleic acids in the cells are more likely to be stained by the fluorescent dye. In other words, for some nucleic acid stains to function as a DNA or RNA stain, the cell membranes need to be damaged.
With specific reference to abnormal cells, paragraph [0043] of the cited Yoshida patent publication (US 2014/0051071) teaches a hemolyzing agent for distinguishably detecting abnormal lymphocytes, atypical lymphocytes and blasts. A hemolyzing agent that contains a nonionic surfactant and substantially no cationic surfactant can be used. Use of the hemolyzing agent allows erythrocytes to be hemolyzed and the cell membranes of normal leukocytes and abnormal mononuclear leukocytes (atypical lymphocytes, abnormal lymphocytes, and blasts) to be damaged. Accordingly, normal leukocytes and abnormal mononuclear leukocytes are more likely to be stained with a fluorescent dye described later in the publication. Paragraph [0049] of that publication also teaches that in addition to the nonionic surfactant a solubilizing agent to sufficiently shrink the hemolyzed erythrocytes so that the hemolyzed erythrocytes form a ghost population that does not adversely affect measurement. From this it appears that the presence of erythrocytes and/or hemolyzed erythrocytes have the potential to adversely affect the measurement.
Paragraphs [0057]-[0058] of Yoshida teach that due to the use of the hemolyzing agent, normal leukocytes and abnormal mononuclear leukocytes are more likely to be stained with a fluorescent dye that will be described, and in addition, abnormal mononuclear leukocytes develop a difference in staining degree, size or other features of abnormal lymphocytes, atypical lymphocytes, and blasts. It is therefore possible based on the fluorescence signal (fluorescence intensity) and the scattered light signal (scattered light intensity) derived from hemocytes to distinguishably detect abnormal lymphocytes, atypical lymphocytes, and blasts in abnormal mononuclear leukocytes. The second reagent is a stain solution for fluorescently staining nucleated cells in a blood sample. A fluorescent dye capable of staining nucleic acid is contained in the second reagent. There is no particular limitation on the fluorescent dye as long as it is capable of fluorescently staining nucleic acid. Such a dye barely stains erythrocytes that do not have nucleic acid, but stains nucleated hemocytes such as abnormal lymphocytes having nucleic acid. The fluorescent dye capable of staining nucleic acid can be suitably selected according to the light irradiated from a light source. Examples of fluorescent dyes capable of staining nucleic acid include propidium iodide, ethidium bromide, ethidium-acridine heterodimer, ethidium diazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, trimethylenebis[[3-[[4-[[(3-methylbenzothiazol-3-ium)-2-yl]methylene]-1,4- -dihydroquinoline]-1-yl]propyl]dimethylaminium].tetraiodide (TOTO-1), 4-[(3-methylbenzothiazol-2(3H)-ylidene)methyl]-1-[3-(trimethylaminio)prop- yl]quinolinium diiodide (TO-PRO-1), N,N,N',N'-tetramethyl-N,N'-bis[3-[4-[3-[(3-methylbenzothiazol-3-ium)-2-yl- ]-2-propenylidene]-1,4-dihydroquinolin-1-yl]propyl]-1,3-propandiaminium tetraiodide (TOTO-3), 2-[3-[[1-[3-(trimethylaminio)propyl]-1,4-dihydroquinolin]-4-ylidene]-1-pr- openyl]-3-methylbenzothiazol-3-ium diiodide (TO-PRO-3), and fluorescent dyes represented by structural formulas (V) to (XVIII) below. Examiner notes that this list and the structural formulas given in the publication are substantially similar to those taught as the second fluorescent dye in instant paragraphs [0120]-[0124] of the originally filed specification.
From this it appears that detection of abnormal lymphocytes, atypical lymphocytes, and blasts in abnormal mononuclear leukocytes using the dyes instantly disclosed as the second fluorescent dye require hemolysis to enable the fluorescent dye to properly function in that detection process.
Finally with respect to the scope of enablement, instant paragraph [0162] of the originally filed specification teaches that the second fluorescent dye is described in US Patent 6,004,816. That patent (Mizukami) teaches that dye as specifically binding to RNA to increase in fluorescence intensity, and a method for classification and counting of leukocytes which uses the reagent, whereby abnormal cells such as immature leukocytes and abnormal leukocytes can be classified and counted easily and highly accurately, and at the same time, classification and counting of normal leukocytes as well as the counting of leukocytes can be performed. Column 2, lines 14-16 teach that the dye is capable of classifying leukocytes into at least 5 populations. Column 2, lines 40-43 teaches mixing a blood sample with a hemolytic agent which lyses erythrocytes in the blood sample to such a degree as not to impede measurement, thereby bringing normal or abnormal cells to a state suitable for staining. Column 3, lines 17-27 teach that the purpose of the mixing step is merely to form pores in the cell membrane of a leukocyte cell to be measured, the pores being of a sufficient size for at least dye molecules to pass through. In other words, it appears that even the preferred second fluorescent dye requires a lysis step to enable it to function as desired.
Thus the scope of the claims are not enabled without a lysis step.
For examination purposes, because the instant claims do not specify what characteristics are used to classify the abnormal cells, examiner will treat the instant claims as including the determination of viable cells as falling within the instant scope of the claims.
Examiner notes that at least acridine orange has different fluorescence characteristics when bound to DNA or RNA (see at least the cited Traganos paper (Journal of Histochemistry and Cytochemistry 1977)) with the DNA fluorescence in the green range and the RNA fluorescence in the red range. Since the RNA fluorescence is in a wavelength range that at least partially overlaps with that taught for the second fluorescent dye by instant paragraphs [0120]-[0124] of the originally filed specification, it is not clear whether acridine orange or any other of the first fluorescent dyes having a similar multicolor fluorescence characteristic combined with the dyes the instant disclosure teaches as the second fluorescent dye creates a problem in classifying any of the abnormal cells due to differences in red fluorescent intensity differences and/or competition with the second fluorescent dye in binding with RNA. While this is an enablement issue, examiner does not have sufficient evidence that this issue would prevent one from being able to use the method of claims 1 or 39 to identify abnormal cells using the fluorescence and scattering data.
Claims 1-39 are rejected under 35 U.S.C. 112(b) 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. With respect to the clarity of the claims, independent claims 1 and 39 require the first fluorescent dye to specifically bind DNA and the second fluorescent dye to specifically bind RNA. If this requirement is interpreted literally, then the first fluorescent dye should not bind with RNA and the second fluorescent dye should not bind with DNA. With regard to the binding behavior of the first fluorescent dye, instant paragraphs [0118]-[0119] of the originally filed specification teach fluorescent dyes with an acridine skeleton as the first fluorescent dye that binds to DNA. Column 4, lines 6-40 of the cited Lefevre Patent (US 7,638,290) teach several stains that are capable of combining specifically with the intracellular ribonucleic acid (RNA) and enhancing its fluorescence once it has combined with the latter. Of relevance to the clarity of the claims are acridine orange, HOECHST 33258 (2'-(4-hydroxyphenyl)-5-(4 methyl-1-piperizinyl)-2,5'-bi-1H-benzimidazole trihydrochloride hydrate), HOECHST 33342® (2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperizinyl)-2,5'-bi-1H-benzimidazole trihydrochloride) and 4',6-diamino-2-phenylindole dihydrochloride (DAPI). In other words, fluorescent dyes which the instant disclosure lists as capable of being the first fluorescent dye and that claims 1 and 39 require to bind specifically to DNA are also known to bind to RNA. That either means that at least these dyes are excluded by the instant claim language or the language of claims 1 and 39 are of a scope that is broader than the literal meaning of the words limiting the first fluorescent dye to specifically bind with DNA.
Additionally, with regard to the clarity of the claims, instant paragraphs [0120]-[0127] of the originally filed specification teach fluorescent dyes having a property of specifically binding RNA of cells. Column 8, line 56 to column 9, line 21 of the cited Vanderlaan Patent (US 5,053,336) discuss staining of DNA and teach several stains for that purpose. Of relevance to the clarity of the claims are the intercalating fluorescent dyes ethidium bromide and propidium iodide. Additionally, paragraph [0092] of the cited Tsuji patent publication (US 2005/0202400) teaches that a standard method for measurement of the DNA amount includes a reagent including propidium iodide. Finally, page 359 of the cited Darzynkiewicz paper (Cytometry 1984) discusses these dyes with respect to binding to and measurement of DNA. In other words, fluorescent dyes which the instant disclosure lists as capable of being the second fluorescent dye and that claims 1 and 39 require to bind specifically to RNA are also known to bind to DNA. That also either means that at least these dyes are excluded by the instant claim language or the language of claims 1 and 39 is of a scope that is broader than the literal meaning of the words limiting the second fluorescent dye to specifically bind with RNA.
Thus there is a real question of whether the scope of the claims covers the dyes listed as the first and second fluorescent dyes in paragraphs [0116]-[0127] of the originally filed specification. For examination purposes examiner will not exclude the above discussed/listed fluorescent dyes/stains from the first or second fluorescent dye scope because they are known to bind to both RNA and DNA.
At this point no art rejection is being made for the following reasons. First, it is difficult to tell what is actually being measured and how it is being combined to determine characteristics that identify abnormal cells. For example, figure 1 of the cited Liegler paper shows how scatter information, first fluorescence information and second fluorescence information can be treated/grouped/displayed to obtain different groupings of peripheral lymphocytes stained with a dye composition using first and second fluorescent dyes within the scope described in instant paragraphs [0116]-[0127] that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. If apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes are considered to be first second and third abnormal cells within the scope of the claims, then this reference appears to anticipate at least claim 1.
Second, the fact that one or more of the dyes listed as either DNA or RNA specific are actually capable of binding to both DNA and RNA merits the question of whether the values applicant measures are unique enough to accurately determine the amount of DNA and/or RNA to make the distinctions that applicant is attempting to make. For example, if one uses acridine orange as the first fluorescent dye, what is its relative binding strength to RNA compared to dye selected as the second fluorescent dye and does the relative amounts and/or amounts of the first and second dyes change the amount of the respective dyes bound to RNA. An additional question is what is the intensity of fluorescence per molecule of acridine orange bound to RNA compared to the intensity of fluorescence per molecule of the second fluorescent dye by bound to RNA. A third question is how many molecules of acridine orange bind to a typical amount of RNA compared to the number of molecule of the second fluorescent dye that bind to a typical amount of RNA. Likewise, similar questions can be asked with respect to ethidium bromide or propidium iodide binding to DNA and how that affects the amount of fluorescence measured from the first fluorescent dye.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. The additionally cited art is directed to cytometric analysis of cellular material including the use of a dual dye staining system for analysis of various aspects of the cells.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Arlen Soderquist whose telephone number is (571)272-1265. The examiner can normally be reached 1st week Monday-Thursday, 2nd week Monday-Friday.
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/ARLEN SODERQUIST/ Primary Examiner, Art Unit 1797
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