The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
The drawings were received on May 1, 2026. These drawings are acceptable.
The disclosure is objected to because of the following informalities: instant paragraph [0047] indicates that the sampling device described therein is not shown. With the above approved drawing changes, that needs to be corrected.
Appropriate correction is required.
With respect to the claims, examiner attempted to determine what constitutes a “large platelet”.
Paragraphs [0053]-[0054] of the instant specification teach that for small platelets (such as platelets with a volume of less than 10 fL), due to small volumes, the platelets are further reduced in volume after being treated with the hemolytic agent. Furthermore, the inventors believe that after a blood sample is hemolyzed, the volumes of platelets are decreased, but the relative sizes are still kept unchanged, that is, the volumes of platelets that are originally large in volume after hemolysis are still large, the platelets that are large in volume can be detected in hemolytic channel through flow cytometry. Instant paragraph [00101] discusses figures 7A-7D teaching that they show data with respect to large platelets with volumes above 10 fL, 12 fL 15 L and 20 fL. There is no distinction relative to the volume and the name that might be used or why one might be interested in a platelet that is larger than any of the stated sizes. There is basis in this paragraph for the size information added to claim 38, but no specific reason for choosing any of the volume thresholds. Thus according to the instant disclosure a large platelet is simply one that is larger in size than the small platelets. For examination purposes, examiner will look for language indicative of a large platelet that has a volume above 20 fL.
Paragraph [0066] of the previously cited Nishimori reference (US 2014/0149938) provides examples of the blood cells to be displayed in an object scattergram. These include, similar to a conventional scattergram, red blood cells (including proerythroblasts which are a premature stage of red blood cells, basophillic erythroblasts, polychromatic erythroblasts, orthochromatic erythroblasts, reticulocytes, and abnormal forms thereof), platelets (including abnormal forms such as large platelets, giant platelets, and the like), and leukocytes (including neutrophils, eosinophils, basophils, lymphocytes, monocytes, atypical lymphocytes, abnormal lymphocytes, large lymphocytes, juvenile cells, and the like). From this it appears that large platelets and/or giant platelets are recognized terms in the art that were normally displayed on scattergrams.
The previously cited Oguni reference (US 2005/0002826) teaches apparatus and method for measuring immature platelets. Paragraph [0024] teaches the finding of a fluorescent dye which produces different staining characteristics between immature platelets and mature platelets, and which is excitable by a laser beam emitted from a semiconductor light source. An assay sample can be prepared using a staining reagent containing the fluorescent dye to detect optical information emitted from the assay sample that can be used to classify and count the immature platelets (this concept includes reticulated platelets). Paragraph [0055] describes figure 7 as an example of the two-dimensional scattergram prepared. This two-dimensional scattergram plots the forward scattered light intensity on the vertical axis, and the fluorescent light intensity on the horizontal axis. The region IP in which the immature platelets appear, and the region MP in which the mature platelets appear, are set. The immature platelets have a greater RNA content within the cell than do mature platelets, and they are larger than the mature platelets. Therefore, the region IP is set at a higher position of forward scattered light and fluorescent light intensities compared to the region MP. From this it is clear that immature platelets such as reticulated platelets are considered larger than mature platelets and could be considered as reticulated platelets for examination purposes. It is also clear that one of ordinary skill in the art is capable of producing a scattergram of fluorescent intensity and forward scattered light intensity and determining areas related to small and large platelets based on size (scatter) and fluorescent intensity (RNA content) for counting purposes.
The previously cited Merchez reference (US 2012/0296570) teaches a method of classifying and flow measuring the refringence of at least two populations of particles present in a fluid. Paragraph [0013] that the ability to distinguish between platelets of large size (macroplatelets) and microcytes (which are red blood cells of very small size), is important in order to avoid making certain errors of diagnosis. Unfortunately, among hematology analyzers other than those based on lasers, there is at present no solution that is simple and effective for avoiding this potential confusion between macroplatelets and microcytes. Paragraph [0089] teaches that the method includes a sub-classification step of classifying normal platelets, activated platelets, microplatelets, and macroplatelets. Since the concept of a "macroplatelet" relates to volume, the sub-classification may be adapted for each laboratory, as with erythrocytes. Paragraph [0090] teaches that the step of classifying events separates populations of leukocytes as constituted in particular by lymphocytes, monocytes, granulocytes, neutrophils, and eosinophilic granulocytes. Paragraphs [0299]-[0300] teach that the platelets are sub-classified in order to distinguish between activated platelets, small platelets, and macroplatelets. This produces four sub-populations of platelets as can be seen in figure 20. If their volume is less than 20 fL, then they are small platelets, and if it is greater than 20 fL, they are macroplatelets. Figures 22A and 22B compare classifications for a case of macroplatelets showing the region of the scattergram that is selected. Thus Merchez shows that one of ordinary skill in the art can identify regions of a scattergram that reflect the presence of large platelets (macroplatelets) having a volume greater than 20 fL and that knowledge of the number of macroplatelets is an important property to determine/measure.
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.
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 38-39, 41-43, 46-49, 51-53 and 56-62 are rejected under 35 U.S.C. 112(a), because the specification, while being enabling for the data processor configured for causing the sampling device to add a fluorescent staining agent and a hemolytic agent as described in the instant disclosure to the reaction cell and missing and incubating the test blood sample, the fluorescent staining agent and the hemolytic agent under the conditions described in the instant disclosure or a method in which the staining and hemolytic reaction are performed with the fluorescent staining agent and a hemolytic agent as described in the instant disclosure under the conditions described in the instant disclosure, does not reasonably provide enablement for use of any fluorescent staining agent, any hemolytic agent under any conditions that they might be applied to cause red blood cell hemolysis. 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. With respect to the wands factors, instant paragraph [0044] gives a simple explanation of the scope of the prior art. In flow cytometric blood cell analyzers, both white cell classification and reticulocyte detection are available, but they cannot be performed in a common measurement channel. The reason is that: in the white blood cell measurement, in order to reduce interference of red blood cells on white blood cells, hemolytic treatment of blood samples is designed to lyse red blood cells into smaller fragments under the effect of hemolytic agents, so as not to affect white blood cell measurement. However, when reticulocytes are detected, it is not expected to destroy reticulocytes in theory. Since hemolytic agents are also capable of lysing reticulocytes, hemolytic treatment of blood samples is not designed in existing flow cytometry reticulocyte measurements.
In support of this, the newly cited Sethu paper describes a continuous flow microfluidic device for rapid erythrocyte lysis from whole blood leukocyte isolation to study inflammation. Leukocytes, however, are sensitive to prolonged exposure to hyper/hypoosmotic solutions, temperature changes, mechanical manipulation, and gradient centrifugation. Even though care is taken to minimize leukocyte activation and cell loss during erythrocyte lysis, it is often not possible to completely avoid it. Most procedures for removal of contaminating erythrocytes from leukocyte preparations are designed for bulk processing of blood, where the sample is manipulated for longer periods of time than necessary at the single-cell level. Ammonium chloride-mediated lysis is the most commonly used method to obtain enriched leukocyte populations but has been shown to cause some activation and selective loss of certain cell types. The leukocyte yield and subsequent activation status of residual leukocytes after NH4Clmediated lysis have been shown to depend on the time of exposure to the lysis buffer. They developed a microfluidic lysis device that deals with erythrocyte removal at nearly the single-cell level, achieving complete lysis of erythrocytes and ~100% recovery of leukocytes where the cells are exposed to an isotonic lysis buffer for less than 40 seconds, after which the leukocytes are immediately returned to physiological conditions. The last full paragraph on page 6247 teaches that NH4Cl-based lysis buffers are widely used and are superior to other lysis solutions and approaches; however, there have been few studies that have examined the actual process. Theoretically, the calculated lysis time for an erythrocyte exposed to an excess NH4Cl-based lysis solution is ~0.1 second, but experimentally, the observed lysis time for a single erythrocyte is ~20 seconds. However, in the macroscale environment, the lysis is diffusion-limited and depends on the concentration of erythrocytes in the lysis solution. During a typical lysis procedure, 1 mL of whole blood is incubated with 15 mL of NH4Cl lysis buffer for 5 min, after which the leukocytes are recovered by centrifugation for another 5 min. It has been shown that incubation with the lysis solution for greater than 10 min resulted in damage to granulocytes and incubation times of 15-20 min were fatal to all leukocytes. There is also evidence to suggest that reduced lysis time can have a favorable effect on cell yield and viability. Table 1 on page 6248 gives a composition for the lysis buffer. The second paragraph of the right column on page 6249 teaches that to characterize erythrocyte lysis and leukocyte recovery for each of the experiments, erythrocytes in the channels were tracked using phase contrast imaging and the cells traveling through the channels were analyzed by taking snapshots at different locations. It was assumed that complete lysis of erythrocytes occurs when greater than 99.5% of the cells were lysed. Cells stained with the SYTO 13 dye were excited using blue fluorescence, and the number of stained cells at the inlet and outlet were counted. The final paragraph on page 6253 of the paper teaches that they developed a microfluidic device for the rapid lysis of erythrocytes where every cell is exposed individually to a lysis solution. Complete lysis of erythrocytes was achieved in ~30 seconds due to significantly reduced diffusion times. All cells were exposed to similar conditions and minimum time of exposure to the lysis buffer resulting in improved cell viability and yield. The output contains leukocytes and is erythrocyte-free (<0.05% original concentration) but contains other contaminants such as reticulocytes (immature erythrocytes), thrombocytes (platelets), and debris. There was no effort to determine what if any change in the reticulocyte numbers occurred in the process.
The newly cited Grimberg paper looked at recovery of reticulocytes from cord blood samples using hypotonic lysis. Exposure of cord blood to hypotonic saline (0.2%) for 5 min selectively lyses the non-reticulocytes resulting in an average 3.6-fold increase in reticulocyte count (see section 2.2 on page 305 for the process). This study shows that this enrichment process does not damage the hemoglobin of the remaining erythrocytes. In section 2.2, it is taught that time and temperature were crucial for successful outcomes. A time course of cord blood exposure to the hypotonic saline indicated that 5 min was the optimal amount of time of exposure to the hypotonic saline solution. After this time point the selectivity of the hypotonic saline for reticulocyte rapidly drops off. At a temperature of 37 °C the method proceeds too quickly and all red blood cells are lysed after 5 min of exposure, and at 4 °C the lysis does not preferentially enrich for reticulocytes but lyses all cell types equally. It is important to note that this enrichment method does not work on normal adult blood. The first full paragraph on page 305 teaches that unlike adult peripheral blood mononuclear cells (PBMCs), flow cytometry of cord blood mononuclear cells has been difficult because of the difficultly in removing contaminating reticulocytes. Even purpose made RBC lysis solutions from Becton–Dickinson, do not lyse all cord blood reticulocytes. This suggested that the osmotic pressure of cord blood reticulocytes is different enough from normocytes to allow for their selective enrichment.
From the above noted teachings of these references it is clear that at least the hypotonic hemolysis agent described by Grimberg is capable of completely lysing reticulocytes in normal adult blood and at least partially lysing the tested cord blood under most conditions. Examiner notes that while the hemolytic agent dose not completely destroy the cord blood reticulocytes under the described optimal conditions for enrichment, there is no indication on what percentage to the reticulocytes are lysed under the optimal treatment conditions. A similar thing can be said relative to the hemolytic agent of Sethu, while there are reticulocytes that survive the treatment conditions described by Sethu, there is nothing that indicates whether all or only a part of the reticulocytes are not lysed. Moreover, Sethu is working with a widely used lysis buffer/agent that is described as superior to other lysis solutions. Additionally both references are clear that one or more of contact time and/or temperature are important in achieving the desired result. In particular with Grimberg, the temperature affects both the selectivity and/or the rate of the lysis. With Sethu the time of contact is critical since longer exposures will cause the white blood cells to also lyse.
An additional factor that the references show is that different types of hemolytic agents don’t produce the same results under the same conditions. Thus the references show that there is a certain amount of experimentation needed for each hemolytic agent. The instantly described hemolytic agents are a small portion of a much larger number of hemolytic agents. Thus any experimentation required (a Wands factor) will be greatly multiplied if one were to search for effective hemolytic agents outside of those that are specifically described.
With respect to enablement, these references show that at least the type of hemolytic agent and conditions such as contact time and contact temperature are critically important to the lysing agent functioning in a manner that will give reliable/usable results. Thus while there is enablement for the instantly described hemolytic agents and contact conditions related to time of contact (incubation) and/or contact temperature, there is not general/generic enablement at the scope covered by the instant claims
No art rejection is being applied against the claims because the art of record fails to teach and/or fairly suggest that mixing a whole blood sample with a hemolytic agent and a fluorescence staining agent would not destroy reticulocytes to the point that passing them through a flow cytometer and recording fluorescence data in combination with first and second (forward and side) scattering data can produce a scattergram using fluorescence data and forward scattering data capable of being used to count/identify reticulocytes along with that same forward scattering data in combination with side scattering data being used to produce a scattergram capable of classifying/counting white blood cells as lymphocytes, monocytes, neutrophils and eosinophils.
Applicant's arguments filed October 23, 2025 have been fully considered but they are not persuasive. In response to the changes made by applicant, the drawing objection has been overcome, the obviousness rejections have been withdrawn, a new objection to the specification has been made based on acceptance of the replacement drawings and a new scope of enablement rejection has been applied against the claims.
With respect to the arguments, since they are directed toward currently withdrawn rejections/objections, the arguments are moot. The arguments are also moot with respect to the new objection and rejection.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. The additionally cited art is related to flow cytometers, measurement of various blood particles and composition of diluents and staining solutions used in platelet and/or reticulocyte analysis apparatus.
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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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Lyle Alexander can be reached on (571)272-1254. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ARLEN SODERQUIST/ Primary Examiner, Art Unit 1797
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