Suspension composition for hematology analysis control

Abstract

A suspension composition for a hematology analysis control particularly useful for preserving relevant detectable characteristics of blood cells for a prolong stability period. The suspension may include at least one polysaccharide, which may include or derive from chitosan and / or chitin, as a stabilizing agent.

Description

(19) *EP004656715A2* (11) EP 4 656 715 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 03.12.2025 Bulletin 2025 / 49 (21) Application number: 25204066.2 (22) Date of filing: 28.07.2017 (51) International Patent Classification (IPC): C12N 5 / 078 (2010.01) (52) Cooperative Patent Classification (CPC): G01N 33 / 5094; C12N 5 / 0634 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 29.07.2016 US 201662368676 P 03.02.2017 US 201762454224 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 17757593.3 / 3 491 381 (71) Applicant: Streck LLC La Vista, NE 68128 (US) (72) Inventors: • HUNSLEY, Brad Papillion, NE 68133 (US) • SCHOLL, John Omaha, NE 68135 (US) (74) Representative: Stolmár & Partner Patentanwälte PartG mbB Blumenstraße 17 80331 München (DE) Remarks: This application was filed on 23.09.2025 as a divisional application to the application mentioned under INID code 62. (54) SUSPENSION COMPOSITION FOR HEMATOLOGY ANALYSIS CONTROL (57) A suspension composition for a hematology analysis control particularly useful for preserving relevant detectable characteristics of blood cells for a prolong stability period. The suspension may include at least one polysaccharide, which may include or derive from chitosan and / or chitin, as a stabilizing agent. EP 4 65 6 71 5 A 2 Processed by Luminess, 75001 PARIS (FR) Description TECHNICAL FIELD

[0001] The teachings relatesgenerally tohematologyanalysis andmoreparticularly to suspensioncompositions foruse in a synthetic control (and resulting control compositions), and a synthetic control, for a semi-automated or automated digital imaging hematology analyzers. BACKGROUND

[0002] For decades, the traditional semi-automated or automated approach to hematology analysis has involved flow based techniques. Blood samples have been aspirated into a hematology analyzer and passed through a detection cell. Relevant characteristics of the blood cells have been measured by light scatter characteristics, electrical characteristics, or otherwise. Based upon detected attributes of the cell, the cell will be classified according to its size and the presence of granular nuclearmaterial. In thismannerbloodcell differentials canbedetermined for sub-populationsofwhitebloodcells.

[0003] In accordance with clinical laboratory standards, performance quality and consistent operation of hematology instrumentshasemployed theuseofcontrols typicallyhavebeenmade frombloodcell sources thatare treated topreserve the relevant detectable characteristics of the cells. Controlmanufacturerswould use either humanor animal blood cells as a blood cell source and process them to achieve and retain a size, density and / or morphology representative of a typical subpopulation of cells, as detected by an instrument detection technique. The respective simulated subpopulations could then be combined in a single control composition, optionally with non-leukocyte blood cell components, such as platelets, reticulated platelets, red blood cells, nucleated red blood cells, reticulocytes, immature reticulocytes, or otherwise.

[0004] Examples of teachings addressing approaches to making simulated blood components are described, without limitation, in US Patent Nos. 4,160,644 and 4,436,821 (teaching simulated platelets); 6,221,668 (teaching simulated reticulocytes, reticulated platelets and nucleated red blood cells); 5,432,089 (teaching simulated reticulocytes, and that suchsimulated reticulocytesmaybederived fromananemicanimal source); 6,723,563 (teachingsimulatednucleated red blood cells); US Published Application No. 20120308985 (teaching simulated immature reticulocytes); The resulting processed cells after transformation from their natural state, would be suspended in a suspension composition adapted to help preserve the processed cells and enable proper function within an instrument. It was a common practice to suspend the cells in amedium that included lipoprotein. This is described, for example, inUnitedStates (US)PatentNos. 5,270,208 and 5,262,327.

[0005] Examples of patents describing the processing of cells from a non-human cell source to form white blood cell subpopulation analogs include US Patent Nos. 5,512,485; see also, US Patent No. 4,704,364. Other examples of teachingsofmakingsimulatedbloodcomponents includeUnitedStatesPatentApplication20030104631;USPatentNos. 6,653,063; 6,514,763; and 5,858,790.

[0006] In recent years, efforts have been devoted toward development of semi-automated or automated hematology analyzers that employ digital imaging to analyze patient blood. Assuring instrument integrity and consistency of results remains a challenge for instrument producers and operators. As with traditional semi-automated or automated hematol- ogy instruments, the digital imaging instruments require regular quality control testing using one or more convenient and accessible control compositions that yield consistent and reproducible results for a prolonged period of time, and well beyond the useful life of fresh whole blood (e.g., fresh human whole blood).

[0007] Due to different detection techniques employed as between traditional semi-automated or automated hematol- ogyanalyzersanddigital imaginghematologyanalyzers, conventional hematologycontrolsarenotnecessarilyusefulwith digital imaginghematologyanalyzers. There is thusaneed for newcompositions for use in a control for semi-automatedor automated digital imaging hematology analyzers. There is a need for a new composition that that provides relatively long termstability of simulatedwhitebloodcells (e.g., at least about3days,7days, 14days,30days,45days, 90daysor longer, such as at least about 105 days) when stored at about 2 to about 10°C. There is a need also for a new composition that allows simulated blood cells to be dispensed (e.g., printed) onto a substrate for delivering the simulated blood cells for semi-automated or automated analysis by digital imaging while on the substrate. SUMMARY

[0008] In general, the present teachings address one or more of the above needs by providing a synthetic control composition for a semi-automated or automated digital imaging hematology analyzers. The present teachings address one ormore of the above needs by providing a unique suspension composition intowhich simulated blood cells (e.g., cells that include simulated nucleated blood cells) can be dispersed to form a synthetic control composition for a semi- automated or automated hematology analyzer, and particularly a digital imaging hematology analyzer. In this regard, the present teachings also relate generally to synthetic compositions for assuring quality control of a digital imaging 2 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 hematology analyzer. The compositions generally will include one or more simulated blood components (which may be derived fromoneormore processed blood cells) and a suspension composition in accordancewith the present teachings. The suspension component is such that stability of the oneormore simulated blood components is prolonged significantly as compared with fresh whole blood (e.g., fresh human whole blood).

[0009] The teachings herein relate as well to a synthetic control composition that includes, in a unique suspension composition, one or any combination of components for simulating a platelet, a reticulated platelet, a red blood cell, reticulocyte, an immature reticulocyte, a nucleated red blood cell, or a simulated leukocyte population or sub-population and is useful as a quality control composition for a digital imaging hematology analyzer. The synthetic control of the teachings may be useful as a quality control composition for a digital imaging hematology analyzer for analyzing a three- part leukocyte differential, a five-part leukocyte differential, and / or an expanded differential leukocyte analysis (also referred to as an expanded differential white blood cell ("dWBC") analysis).

[0010] It can be seen that the synthetic control composition of the teachings may include simulated leukocytes (which maybederived fromahumanor other source, asdiscussedherein) providedasan individual subpopulationof leukocytes, and / or a collection of cells capable of differentiation into at least the three and / or the five traditional subpopulations of leukocytes, and / or the simulated leukocytes may be suitable for providing an expanded differential white blood cell ("dWBC") analysis and for total white blood cell count or cell count for any white blood cell sub population.

[0011] With attention now to certain other generalities about the present teachings, it can be said that the teachings herein relate generally to a composition adapted for use in assuring quality control of a digital imaging hematology analyzer, including a suspension medium with which at least one simulated nucleated blood component is mixed in a predetermined amount, the suspension medium including a stabilizing agent capable of preserving relevant detectable size and morphology, including detectable nuclear morphological characteristics of the nucleated blood component (including any native nuclear cytoplasm granules), when stored at about 2 to about 10°C for a period of at least 3 days, 7 days, 14 days, 30 days, 45 days, 90 days or longer, such as at least about 105 days) from the time when the at least one simulated nucleated blood component is initially mixed with the suspension medium.

[0012] Thecontrol composition inwhich thesuspensioncompositionof the teachings isusefulmaybeasynthetic control composition for simulating one ormore components of blood (e.g., humanwhole blood). The control composition inwhich the suspension composition of the teachings is useful may be a control composition for simulating cells of a leukocyte population of whole blood (e.g., simulated nucleated blood cells to resemble a leukocyte population or one or more subpopulations). Examples of a leukocyte population of whole blood for use in a control composition with the present suspension composition include a three-part leukocyte population of whole blood (e.g., the control may be to simulate three leukocyte subpopulations of whole blood (namely, neutrophils, lymphocytes andmonocytes)), a five-part leukocyte population of whole blood (e.g., the controlmay be to simulate the traditional five leukocyte subpopulations ofwhole blood (namely, neutrophils, eosinophils, basophils, lymphocytes, andmonocytes)), and / or an extended leukocyte population of whole blood (e.g., the control may include further subpopulations of one or more of the traditional five leukocyte subpopulations of whole blood). An example of another simulated nucleated cell component is a nucleated red blood cell. The suspension composition of the present teachings are also useful to preserve simulated reticulocytes, an immature reticulocyte fraction, or both.

[0013] The suspension compositionmay be employed in a synthetic control composition. The suspension composition may be employed in a synthetic control composition in combination with simulated blood cells (e.g., simulated nucleated blood cells, such as a simulated leukocyte population or sub-population of whole blood and / or a simulated nucleated red blood cell). A simulated leukocytepopulationor subpopulationofwholeblood, for thepresent teachings,maybederivedat least partially, or entirely from leukocytes of human whole blood. The simulated leukocyte population or subpopulation of whole blood may include cells that have been treated in a manner to stabilize their respective cell membranes so that the cells remain substantially intact for a period of time that is longer than cells that are not stabilized. It is possible that all simulated leukocytes of a leukocyte population, including the respective subpopulations, are treated simultaneously (e.g., according to a sequence of one or more partial or complete fixing and lysing steps) to provide the simulated leukocytes.

[0014] The suspension composition may be employed in a control composition, in combination with one or more components for simulating a platelet, a reticulated platelet, a red blood cell, reticulocyte, an immature reticulocyte, a nucleated red blood cell, or any combination thereof. For example, the suspension composition may be employed in a control composition in combination with a simulated nucleated red blood cell prepared from a non-human source (e.g., nucleated blood cells of a bird such as a turkey, a fish, or a reptile such as an alligator). For example, it may be possible to provide a source of blood cells suitable for simulating a nucleated red blood cell. Provided blood cells may individually include a membrane enclosing a nucleus and cytoplasm. The membrane may be stabilized to retain the nucleus and cytoplasm. For example, there may be a step of lysing the cells that are provided (e.g., with a solution including saponin) and then fixingwith analdehyde (e.g., glutaraldehydeand / or formaldehyde). It is alsopossible that no lysing stepwouldbe utilized prior to cell fixation.

[0015] The amounts of simulated cells in the suspension composition generally are predetermined; for example, the amounts may be known amounts for simulating normal amounts in whole blood and / or abnormal amounts). The 3 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 suspensioncompositionmaybeuseful in astand-alonecontrol composition, inwhich typically asinglesimulatedbloodcell component is employed.

[0016] In contrast with prior control compositions, the control composition of the present teachings need not necessarily employa lipoprotein to helpassureproper characterizationanddifferentiationof simulated leukocyte cells into their proper subpopulations. The present teachings make use of the recognition that certain stabilization agents can be mixed in a suspension composition with a volume of simulated white blood cells, and for a prolonged period (e.g., at least about 3 days, 7 days, 14days, 30days, 45days, 90daysor longer, suchasat least about 105days,whenstoredat about 2 to about 10°C) from the time of mixing, simulated cell components (including the simulated leukocytes) retain relevant detectable size and morphology, including detectable nuclear morphological characteristics of the cell and its nuclear matter, including native nuclear cytoplasm granules. However, it is also possible that lipoprotein may be utilized.

[0017] According to one general aspect of the teachings applicable to all embodiments, there is contemplated a suspension composition for simulated leukocytes (e.g., cells that have similar detectable characteristics, such as size, a nucleus morphology and / or morphology of other native nuclear cytoplasm granules, of one or any combination of sub- populationsofwholeblood)of ahematologyanalyzer control formulation.Thesuspensioncomposition isespecially useful for a control for a semi-automated or automated digital imaging analyzer. The suspension composition is adapted to retain the detectable characteristics of the simulated blood cells (e.g., simulated nucleated cells such as nucleated leukocytes and / or nucleated red blood cells) over a prolonged period of storage. The suspension compositionmay include a buffered aqueous solution.

[0018] As to all embodiments, the suspension composition may include at least one stabilizing agent (e.g., one that includes at least one polysaccharide, such as (without limitation) a nitrogen-containing polysaccharide) having a polymerization degree ranging from greater than one to about 100). The at least one stabilizing agent may be present in an amount sufficient for preserving stability of the detectable size and morphological characteristics of the simulated simulated blood cells (e.g., simulated nucleated cells such as nucleated leukocytes and / or nucleated red blood cells) for a period of at least about 3 days (e.g., at least about 3 days, 7 days, 14 days, 30 days, 45 days, 90 days or longer, such as at least about 105 days) when stored at about 2 to about 10°C), from the time of suspending the simulated cells.

[0019] Generally, the suspension composition may be adapted (as to all embodiments) for use in digital imaging hematology instrument that creates and analyzes an image, such as by applying one or more automated or semi- automated analytical techniques, after a sample has been dispensed onto a substrate. For instance, upon mixing the suspension compositionwith the simulated leukocytes, the resultingmixture is capable of dispensing through a nozzle for delivery to a substrate for analysis by a digital imaging hematology analyzer. The suspension composition may be adapted, uponmixing with the simulated blood cells (e.g., simulated nucleated cells such as nucleated leukocytes and / or nucleated red blood cells), for dispensing through a nozzle (e.g., a capillary or other tube) for delivery by printing to a transparent substrate (e.g., a glass or polymeric slide) and subsequent analysis by a semi-automated or automated digital imaging hematology analyzer, without any material damage (e.g., damage to excess of five percent (10%) by number of total simulated cells) to the simulated cells. It is possible that the control composition may be delivered into a cassette or cartridge device which may provide digital images for identifying cell populations.

[0020] In general, as to all embodiments, the aqueous buffered solution of the suspension composition may include at least one buffering agent.

[0021] The aqueous buffered solution may include at least one dispersion agent for reducing aggregation of the simulated blood cells (e.g., simulated nucleated cells such as nucleated leukocytes and / or nucleated red blood cells) as compared with the aqueous buffered solution without the dispersion agent.

[0022] The suspension composition may have a pH ranging from about 6 to about 8.

[0023] Theat least onestabilizingagentmay includeanorganic compoundhavingat least oneglycosidic linkage.Theat least one stabilizing agent may include a compound that includes an amine moiety and a carbohydrate (e.g., glucose and / or dextrose) moiety.

[0024] Theat least one stabilizing agentmay include at least one aminopolysaccharide having apolymerization degree ranging from greater than one to about 100 (e.g., from greater than about 5 to about 40). The at least one stabilizing agent may include or consist of an oligosaccharide (or a derivative of an oligosaccharide) having a weight average molecular weight (measured by high performance liquid chromatography, which may be further verified by comparison with commercially available standards)) from about 100 to about 15,000 daltons (Da), from about 250 to about 10,000 Da, about or even about 1000 to about 3000 Da.

[0025] Theat least onestabilizingagentmay includeaglucosamine,oraderivative thereof. Itmay includeoneormoreof a chitosan and / or chitin, a salt of a chitosan and / or chitin, and / or some other derivative of a chitosan and / or chitin.

[0026] The at least one stabilizing agent may be in a polymeric form. The at least one stabilizing agent may be in a salt form (e.g., a salt of a chitosanand / or chitin). Illustrative salts includeoneor any combinationof a citrate, amalate, a lactate, an acetate, a formate, a glyoxylate, a pyruvate, an ascorbate or glycolate.

[0027] Generally applicable to all embodiments, the at least one stabilizing agent may be present in an amount up to about ten percent (15%) (e.g., up to about ten percent (10%) or about seven percent (7%)) of the suspension composition. 4 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 The at least one stabilizing agent may be present in an amount up to about ten percent (10%) (e.g., up to about seven percent (7%)or about fivepercent (5%)of a resulting control compositionadmixture including the suspension composition and the simulated cells.

[0028] Unlike certain control compositions of the prior art, the suspension composition and any resulting control composition employing the suspension composition of the general teachings herein may be substantially free of any added lipid (e.g., lipoprotein), any glycoprotein or both.

[0029] The teachings herein also contemplate a blood control composition (e.g., leukocyte and / or nucleated red blood cell-containing control) composition adapted for use in a semi-automated or automated digital imaging hematology analyzer comprising the suspension composition of the teachings, aswell as the use of the composition. For instance, the teachings envision a method of using the suspension composition including a step of dispensing the suspension composition onto a substrate, andat least partially evaporatingwater from theaqueous buffered suspension composition. Thereafter, a digital image can be made. For instance, when cells are deposited onto a substrate with the suspension composition, a digital image may be made of the cells. The image may be analyzed. For instance the image may be analyzed by a computer implemented technique.

[0030] Otherbenefitsandadvantagesof the teachingswill beunderstoodupon reviewof the remaining teachings,which provide additional details. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Fig. 1 isamicrograph illustratingcellsof freshhumanwholeblood,asviewedbyopticalmicroscopy (oil immersion lens at 100x), in a form as would be detectable by a digital imaging hematology analyzer. Fig. 2 is a micrograph illustrating simulated leukocytes cells in a freshly prepared suspension composition of the present teachings as viewed by optical microscopy (oil immersion lens at 100x), in a form as would be analyzed by a digital imaging hematology analyzer. Fig. 3a is anenlargedmicrograph illustrating respectiveexamplesof simulate leukocyte subpopulation cells andother simulated cell components in a suspension composition of the present teachings as viewed by opticalmicroscopy (oil immersion lens at 100x), in a form as would be analyzed by a digital imaging hematology analyzer. Fig. 3b is an enlarged micrograph illustrating respective examples of leukocyte subpopulation cells and other simulated cell components after storage at about 2 to about 10°C for 105 days in a suspension composition of the present teachings as viewed by opticalmicroscopy (oil immersion lens at 100x), in a formaswould be analyzed by a digital imaging hematology analyzer. Fig. 4a is a micrograph to illustrate an example of simulated leukocyte cells in a suspension medium including a stabilizing agent of the present teachings after about threeweeksas viewedby opticalmicroscopy (oil immersion lens at 100x), in a form as would be analyzed by a digital imaging hematology analyzer. Fig. 4b is a micrograph to illustrate an example of simulated leukocyte cells of the present teachings in a suspension medium as in Fig. 4a, but absent any stabilizing agent of Fig. 4a after about three weeks as viewed by optical microscopy (oil immersion lens at 100x), in a form as would be analyzed by a digital imaging hematology analyzer. Fig. 5 is a micrograph to illustrate an example of a prior art hematology control, absent any stabilizing agent of the present teachings, as viewed by optical microscopy (oil immersion lens at 100x), in a form as would be analyzed by a digital imaging hematology analyzer. DETAILED DESCRIPTION

[0032] The explanations and illustrations presented herein are intended to acquaint others skilled in the art with the teachings, its principles, and its practical application. Those skilled in the art may adapt and apply the teachings in its numerous forms, as may be best suited to the requirements of a particular use. Accordingly, the specific embodiments of the present teachings as set forth are not intended as being exhaustive or limiting of the teachings. The scope of the teachings should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes. Other combinations are also possible as will be gleaned from the following claims, which are also hereby incorporated by reference into this written description.

[0033] This application claims the benefit of the filing date of U.S. Provisional Application Serial Nos. 62 / 368,676, filed July 29, 2016 and 62 / 454,224, filed February 3, 2017, the contents of these applications being hereby incorporated by reference herein for all purposes.

[0034] Unlessotherwisestated, concentrationsareexpressed inweight / volumepercentages (w / v%).Thus, to illustrate, 5 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 1%of an ingredient in a liquidmediumwould refer to 1gramof the ingredient in 100milliliters of the liquid.Unlessotherwise stated, references herein to "whole blood" and / or "blood cells", include human whole blood. Though the teachings herein are particularly applicable in human whole blood analysis, they are not so limited, and may have application in veterinary applications aswell. The phrase "digital imaging hematology instrument" refers to automated hematology analyzers of an automated or semi-automated type that employ computer implemented analysis of a digital image of a sample (e.g., a computer may analyze the sample to ascertain cell size, cell morphology, count cells, and / or classify cells, and may also output resulting data and / or images for independent human analysis). An example of such analysis technology is exemplified by the Cobas m511™, by Roche Diagnostics, which employs what is referred to as Bloodhound™ analysis technology. It is believed that such technology is described in one or more of U.S. Patent Application Nos. 20090269799; 20110014645; 20130070077; 20130077085; and WO / 2013 / 016038.

[0035] Thephrase "detectable characteristics" refers to oneormore characteristics (e.g., a physical characteristic, such as size and / or morphology) that are detectable by a hematology instrument, whether a digital imaging hematology instrument or otherwise.

[0036] In general, the teachings herein contemplates a suspension composition for preserving the stability of simulated blood cells of a hematology control composition. The teachingsmore particularly contemplates a suspension composition for preserving the stability of simulated blood cells, so that the cells exhibit and retain relevant detectable characteristics, such as size, and / ormorphology.One particular advantage of the teachings is the ability of the suspension composition to preserve the stability nuclear morphological characteristics (including morphology of native nuclear cytoplasm granules) for a period substantially longer than fresh human whole blood. The suspension composition teachings are useful to prepare a suspension of a control composition for assuring quality of a hematology analyzer (e.g., a digital imaging hematology analyzer).

[0037] In general, the suspension composition is useful for and may be part of a control composition that include the suspension and simulated blood cells (e.g., simulated nucleated cells such as nucleated leukocytes and / or nucleated red blood cells). Such control composition and methods of using the controls are part of the teachings herein as well.

[0038] Determination of whether relevant detectable characteristics are retained can be done by optical examination (e.g., including the use of a digital imaging device for outputting an image) of a statistically significant number of samples, with analysis to identify material deviations from native morphological attributes of the type of cell under consideration. Indicators ofmaterial deviations can employ opticalmeans to identify. It may include a comparative study of a samplewith data (which may include images) about known characteristics of the sample material in its normal freshly prepared state. The skilled person likewise would recognize that relevant detectable characteristics have not been attained, or are no longer retained (e.g., a stability periodhasbeenexceeded)whenasample intendedyields cell counts that deviate from the intended values in excess of at least ten percent (10%), or there is some other evidence indicative of an inability to differentiate cells; when nucleated blood cells present identifiable surface cracks in images at a magnification of at least 100X (e.g., when viewed by optical microscopy with an oil immersion lens); when cellular cytoplasm appears de- granulated and not readily identifiable in images at a magnification of at least 100X (e.g., when viewed by optical microscopy with an oil immersion lens), and / or when one ormore nuclei sizes deviate from their normal well known sizes.

[0039] It should thusbe recognized, that thephrase "simulatedbloodcells" is usedhereinnot only to refers tocells froma non-human source that are adapted to resemble blood cells from a human (or those of another species) in relevant detectable characteristics by a hematology analyzer (e.g., a digital imaging analyzer). The phrase "simulated blood cells" also refers to blood cells that are true human blood cells. Thus, a source of simulatedwhite blood cells, in accordancewith the teachings hereinmaybehumanwhite blood cells.Moreover, any of the simulated blood cells that are suspended in the suspension compositionmaybe cells derived fromhumanwholebloodand / or non-humanwhole blood source (e.g., blood from a source selected from avian blood, fish blood, reptilian blood, mammalian blood or otherwise.

[0040] The suspension composition may be employed in a control composition, alone or in combination with simulated blood cells, which may include simulated leukocytes or other simulated cell components of whole blood (e.g., simulated nucleated cells such as nucleated leukocytes and / or nucleated red blood cells). A simulated leukocyte population or subpopulation of whole blood may be derived at least partially, or entirely from leukocytes of human whole blood. The simulated leukocyte population or subpopulation of whole blood may include cells that have been treated in a manner to stabilize their respective cell membranes so that the cells remain substantially intact for a period of time that is longer than cells that are unstabilized. It is possible that all simulated leukocytes of a leukocyte population, including the respective subpopulations, are treated simultaneously to provide the simulated leukocytes. For example, a source of leukocytes (e.g., a leukocyte pack from a blood bank) may be provided and the leukocytes contacted with a stabilizer to partially stabilize the cell membranes of the leukocytes. The source is then lysed to eliminate red blood cells. The leukocytes may then be further stabilized prior to combining them with the suspension.

[0041] In general, the present teachings provide a unique suspension composition into which simulated blood cells can be dispersed to form a control composition. In contrast with prior control compositions, the control composition of the present teachings need not necessarily employ a lipoprotein to help assure proper characterization of simulated leukocytes and / or differentiation of the simulated blood cells into their proper subpopulations. 6 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55

[0042] Thepresent teachingsmake useof the recognition that certain stabilization agents can bemixed in a suspension composition with a volume of simulated blood cells (e.g., for simulation one or more subpopulations of white blood cells), and for a prolonged stability period (e.g., at least about 3, at least about 7, at least about 14, at least about 30, at least about 45, at least about 90 days or longer, e.g., at least about 105 days, while maintained at a temperature of about 2 to about 10°C) from time of mixing, the simulated blood cells (e.g., simulated leukocytes, and any other simulated blood cell component such as a nucleated red blood cell component) retain relevant detectable characteristics such as size and / or nuclear morphological characteristics (including morphology of native nuclear cytoplasm granules).

[0043] According to one general aspect of the teachings applicable to all embodiments, there is contemplated a suspension composition for simulated blood cells of a hematology analyzer control formulation, which may be a digital imaging hematology analyzer. The simulated blood cells may be cells from a suitable source that have been processed to attain and / or preserve characteristics detectable by a hematology analyzer, such as a digital imaging hematology analyzer. For instance, the characteristics that are attained and / or preserved may be size, a nucleus morphology and / or morphology of other nuclear cytoplasm granules.

[0044] The teachings herein have application for suspending and preserving stability of simulated blood components (e.g., blood cells) in a control composition. For example, the teachings have application for suspending and preserving stability of simulated leukocytes of a control composition. The simulated leukocytes (whichmay be derived from a human or other source, as discussed above)may be provided as an individual subpopulation of leukocytes, and / or a collection of cells capable of differentiation into at least the threeand / or the five traditional subpopulations of leukocytes. The simulated leukocytes may be suitable for providing an expanded differential white blood cell ("dWBC") analysis.

[0045] A control in accordance with the teachings for an expanded dWBC analysis may include (e.g., in addition to the traditional 3or 5subpopulations) components that simulateoneormorecell populations thatmay includeblasts, immature granulocytes, atypical lymphocytes (types I, II and III), myeloid precursors (myeloblast, promyelocyte, myelocyte and metamyelocyte) and or lymphocyte subsets (T-Cells and B-Cells).

[0046] The suspension composition is adapted to retain the detectable characteristics of the simulated blood cells (e.g., white blood cells) over a prolonged period of storage. For example, the suspension composition (whether used for simulated white blood cells and / or other blood cells is formulated to preserve relevant detectable characteristics, such as size and / or nuclear morphological characteristics (including morphology of native nuclear cytoplasm granules) of the simulatedblood cells, for aprolonged stability period (e.g., at least about 3, at least about 7, at least about 14, at least about 30, at least about 45, at least about 90 days or longer, e.g., at least about 105 days) when stored at about 2 to about 10°C from time of mixing with the suspension composition.

[0047] Throughout the prolonged stability period, the nucleated cells (e.g., the simulated leukocytes (and / or any other simulated cell components such as simulated nucleated red blood cells) will resemble to a detector of a hematology analyzer, and particularly a digital imaging hematology analyzer (e.g., without limitation, a COBASm511™ analyzer from Roche) the blood cell it is intended to simulate. Thus, the simulated blood cell is capable of being subjected to sample processing by a digital imaging hematology instrument, during which the cell may be deposited on a slide (such as by a printing operation), stained, and imaged in a manner suitable for computer implemented image analysis.

[0048] The suspension composition may be isotonic. The suspension composition may include a buffered aqueous solution. The suspension composition may include amixture of cell stabilizers. The suspension composition may include an antimicrobial. The suspension composition may include one or more agents for maintaining a predetermined pH.

[0049] As to all embodiments, the suspension composition may include at least one stabilizing agent (e.g., one that includes at least one polysaccharide having a polymerization degree ranging from greater than one to about 100, such as (without limitation) a nitrogen containing polysaccharide). The at least one stabilizing agent may be present in an amount sufficient for preserving stability of the detectable morphological characteristics of the white blood cells for a period of at least about 3 days (e.g., at least about 7, at least about 14, at least about 30, at least about 45, at least about 90 days or longer, e.g., at least about 105 days), after being stored during such period at about 2 to about 10°C, upon suspending the simulated white blood cells.

[0050] Generally, the suspension composition may be adapted (as to all embodiments) for use in digital imaging hematology instrument that creates and analyzes by a computer implemented technique an image of a sample that has been dispensed onto a substrate. For instance, upon mixing the suspension composition with the simulated leukocytes, the resulting mixture is capable of dispensing through a nozzle for delivery to a substrate for analysis by a digital imaging hematology analyzer. The suspension compositionmay be adapted, uponmixing with the simulated white blood cells, for dispensing through a nozzle (e.g., a capillary or other tube) for delivery by printing to a transparent substrate (e.g., a glass or polymeric slide) and subsequent analysis by a digital imaging hematology analyzer, without anymaterial damage (e.g., damage to excess of five percent (10%) by number of total simulated blood cells) to the simulated blood cells.

[0051] The at least one stabilizing agent may be present (as to all embodiments) in an amount sufficient for preserving stability of the detectable morphological characteristics of the white blood cells for a period of at least 30 days, after being stored during such period at about 2 to about 10°C, upon suspending thewhite blood cells.More specifically, as applicable to the teachings in general, the at least one stabilizing agent may function to retain intact relevant detectable character- 7 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 istics such as size and / or nuclear morphological characteristics (including morphology of native nuclear cytoplasm granules) of one or more of any simulated nucleated blood cell component of a control composition. Thus, the size and shape of a nucleus of the simulated blood cell be preserved substantially as it would be in its native state in fresh whole blood. It is also possible that the size, shape and / or amount of other nuclear matter (e.g., cytoplasm) may be preserved substantially as it would be in its native state in fresh whole blood.

[0052] The at least one stabilizing agent may be part of a stabilization mixture. Such stabilization mixture may include two, three, four, fiveormore ingredients.Suchstabilizationmixturemay include twelve, ten, eight or fewer ingredients. The ingredients of the stabilization mixture may be in an amount effective for stabilizing one or more aspects of a simulated blood cell (e.g., a simulated leukocyte or any other simulated blood cell component, such as a platelet, a reticulated platelet, a redbloodcell, reticulocyte, an immature reticulocyte, anucleated redbloodcell, or anycombination thereof). For example, oneormoreof the ingredientsmaybe for stabilizing amembrane, for stabilizing nuclearmaterial, for stabilizing a nucleic acid, for stabilizing cytoplasm, or otherwise).

[0053] In general, as to all embodiments, the aqueous buffered solution of the suspension composition may include at least one buffering agent. Itmay include at least one antimicrobial. Theaqueous buffered solutionmay include at least one dispersion agent for reducing aggregation of the simulated white blood cells as compared with the aqueous buffered solution without the dispersion agent. The suspension composition may have a pH ranging from about 6 to about 8.

[0054] In general, as to all embodiments, with regard to the at least one stabilizing agent, it may include at least one polysaccharide, such as is set forth in the following. The at least one stabilizing agent may be a nitrogen-containing polysaccharide. The at least one stabilizing agent may be a derivative of glucose. It may include one or more glucose moieties. However, the at least one stabilizing agent may be substantially free of simple glucose (i.e., that represented by the formula C6H12O6); that is, the suspensionmediummay include less than about 10%, or less than about 5%glucose of the suspension medium.

[0055] For example, the at least one stabilizing agent may include at least one amino polysaccharide having a polymerization degree ranging from greater than one to about 100 (e.g., from greater than about 5 to about 40). The at least one stabilizing agent may include or consist of an oligosaccharide (or a derivative of an oligosaccharide) having a weight averagemolecularweight (measured byhighperformance liquid chromatography,whichmaybe further verifiedby comparisonwith commercially available standards)) fromabout 250 toabout 10,000daltons (Da), fromabout 250 to about 10,000 Da, or even about 1000 to about 3000 Da.

[0056] Theat least onestabilizingagentmay includeaglucosamine,oraderivative thereof. Itmay includeoneormoreof a chitosan, a salt of a chitosan or some other derivative of a chitosan.

[0057] The at least one stabilizing agent may be in a polymeric form. The at least one stabilizing agent may be in a salt form (e.g., a salt of achitosan). Illustrative salts includeoneoranycombinationof acitrate, amalate, a lactate, anacetate, a formate, a glyoxylate, a pyruvate, an ascorbate or glycolate.

[0058] Byway of example, the at least one stabilizing agent may include one or any combination of D‑(+)‑glucosamine, N-acetyl-D-glucosamine, chitosan acetate, chitosan lactate, chitosan oligosaccharide, chitin, carboxyl methyl chitosan, a derivative of any of these listed agents, or any combination of the same.

[0059] Generally applicable to all embodiments, the at least one stabilizing agent may be present in an amount up to about fifteen percent (15%) (e.g., up to about ten percent (10%) or about seven per cent (7%)) about ten percent (10%) or about seven per cent (7%)) of the suspension composition. The at least one stabilizing agentmay be present in an amount up to about ten percent (10%) (e.g., up to about seven percent (7%) or about five percent (5%) of a resulting control composition admixture including the suspension composition and the white blood cells. Generally applicable to all embodiments, the at least one stabilizing agent along with any other ingredients functional for preserving stability of a simulated blood cell (i.e., the total functional ingredients of the stabilizing mixture), may be present in amount up to about twenty five percent (25%); twenty two percent (22%); or about eighteen (18%) (e.g., up to about fifteen percent (15%) or about ten per cent (10%)) of the suspension composition. The total functional ingredients of the stabilizingmixturemay be present in anamount of at least about onepercent (1%) (e.g., at least about sevenpercent (3%)or about fivepercent (5%)) of a resulting control composition admixture including the suspension composition and the white blood cells.

[0060] Unlike certain control compositions of the prior art, the suspension composition and any resulting control composition employing the suspension composition of the general teachings herein may be substantially free of any added lipid (e.g., lipoprotein), any glycoprotein or both. For example, it may have less than 0.3 percent (e.g., less than about 0.1 percent) of a lipid and / or glycoprotein. Alternatively stated, to the extent any lipid (e.g., lipoprotein), any glycoprotein or both are employed in a suspension composition in accordancewith the present teachings, such lipid (e.g., lipoprotein), glycoprotein or both is employed in an insufficient amount tomaterially alter the stability characteristics of any of the resulting blood components of the resulting control composition during the stability period. For instance, the amount is an amount at which detectable nuclear morphological characteristics of the nucleated blood component (including any native nuclear cytoplasmgranules) exhibit less than a 10%, or even less than a 5%, variation in detected cell counts when analyzed by a digital hematology analyzer as comparedwith a control having no such lipid (e.g., lipoprotein), glycoprotein or both. 8 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55

[0061] Though the suspension composition may be substantially free of any lipoprotein, it may optionally include lipoprotein. For instance, it may include a lipoprotein in a suitable amount for assuring proper differentiation of white blood cells added to the suspension composition. The suspension composition may include, or it may be free of an antioxidant (e.g., in anamount sufficient to prevent cell lysis during the stability period of a control inwhich the suspension composition is used).

[0062] The suspension compositionmay include at least one cellular aggregation inhibition ingredient, ormay be free of any cellular aggregation inhibition ingredient. The suspension composition may include a surfactant (e.g., in an amount sufficient to effect a surface energy characteristic of any of the simulated blood cells), or it may be free of any surfactant. Examples of surfactants can be found in U.S. Patent No. 5.250,438, (see, e.g., column 3), incorporated by reference.

[0063] In general, as to all embodiments, the aqueous buffered solution of the suspension composition may include at least one buffering agent. For example, itmay be zwitterionic buffering agent. The at least one buffering agentmay include a sulfonic acid moiety. The buffering agent may be selected from one or any combination of 3-Morpholinopropane‑1- sulfonic acid (MOPS); 2‑[[1,3-dihydroxy‑2‑(hydroxymethyl)propan‑2-yl]amino]ethanesulfonic acid (TES); or2‑[4‑(2-hy- droxyethyl)piperazin‑1-yl]ethanesulfonic acid (HEPES). The at least one buffering agent may be present in an amount of up to about 7 percent, up to about 5 percent or up to about 3 percent of the suspension composition. The at least one buffering agent may be present in an amount of greater than about 0.5 percent or greater than about 1 percent of the suspension composition.

[0064] Thesuspension compositionmay include at least one antimicrobial. The at least one antimicrobialmay bepart of the aqueous buffered solution or a separate ingredient. The at least one antimicrobial may be organic or inorganic. The at least one antimicrobial may include a biocide, a microbe growth inhibitor, and / or a microbe reproduction inhibitor. The at least one anti-microbial may be a bacteriostatic ingredient (e.g., one that inhibits cytochrome oxidase in gram negative bacteria). The at least one anti-microbial may be an anti-fungal ingredient. The at least one anti-microbial may be an anti- yeast ingredient. Examples of suitable antimicrobials include one or more antimicrobials selected from chloramphenicol, sodium azide, neomycin sulfate, Rifampicin minocycline, ciproflaxin, doxycycline, sulfasalazine or the like. The at least one antimicrobial may be present in an amount of up to about 5%, up to about 3% or up to about1 % percent of the suspension composition.

[0065] The suspension composition may have a pH ranging from about 6 to about 8. Suitable amounts of neutralizing agents may be employed in the suspension composition. For example, a relatively mild acid such and / or a relatively mild base such as sodium hydroxide may be titrated into the suspension composition as needed to achieve the desired pH level.

[0066] As gleaned from the above, it is envisioned that the suspension composition may include one or more inorganic compounds. The inorganic compoundmay include ametallic atom or cation, such as an atom or cation of an alkali metal, an alkaline earth metal, a transition metal, or any combination thereof. For example, it is possible that the resulting suspension composition will have one or more metal atoms or cations selected from magnesium (Mg), calcium (Ca), sodium (Na), potassium (K), lithium (Li), iron (Fe), or otherwise. The amount of the metal atoms or cations may be quantifiedasaproportion relative to thepolysaccharide of the suspension composition. For example, theamount of atoms or cations byweight relative to the polysaccharidemay range fromabout 0.1:1 to about 2:1 (e.g., about 0.3:1 to about 1:1).

[0067] To the extent not covered in the above, the suspension composition may include one or more other ingredients, including one or any combination of an alcohol, a surfactant; a protease inhibitor; serumalbumin protein; an anticoagulant (e.g., ethylenediaminetetraacetic acid "EDTA"), a gelatin, an aldehyde (e.g., glutaraldehyde and / or formaldehyde), a polyol (e.g., polyethylene glycol), a cellulosic agent, an antioxidant, a protein, a blood serum fraction, plasma, amino acid precursor, a cholesterol (e.g., a lipoprotein), or any combination of the above.

[0068] Ingeneral,waterused for theaqueouscompositionshereinmaybedistilledwater, deionizedwater, filteredwater, anycombination thereof oranother sourceofwater that is freeofdetectableamountsof contaminants thatwouldmaterially impact performance of the suspension composition.

[0069] The teachings herein also contemplate a blood control composition (e.g., a white blood cell control composition) adapted for use in a digital imaging hematology analyzer comprising the suspension composition of the teachings, aswell as the use of the composition. For instance, the teachings envision a method of using the suspension composition including a step of dispensing the suspension composition onto a substrate, and at least partially evaporating water from the aqueous buffered suspension composition. Thereafter, a digital image can be made. For instance, when cells are deposited onto a substrate with the suspension composition, a digital imagemay bemade of the cells. The imagemay be analyzed.

[0070] Turning now to the accompanying Figs. 1, 2, 3a, 3b, 4a, 4b, and 5 various benefits and advantages of the teachings canbe further gleaned. Except as otherwise described, the imagesare believed representative of compositions thatwould result usinga suspensionmedium, generally, of thepresent teachings, andparticularly the suspensionmedium described below in Example 1. The images are believed representative of compositions that would result from simulated cell components that include simulated leukocytes, wherein the simulated leukocytes are derived fromhumanblood cells. Similar results are also expected when the simulated leukocytes are derived from non-human blood cells. 9 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55

[0071] Fig. 1 is a micrograph illustrating cells of fresh human whole blood, as viewed by optical microscopy (oil immersion lens at 100x), in a form as would be detectable by a digital imaging hematology analyzer. Examples of the cellular components are labeled for reference purposes. Upon draw, the respective cellular components of the fresh human whole blood substantially identically resemble the cellular components as would be circulating within person from whom the blood was drawn.

[0072] Fig. 2 is amicrograph illustrating simulated leukocytes cells in a freshly prepared suspension composition of the present teachings as viewed by optical microscopy (oil immersion lens at 100x), in a form as would be detectable by a digital imaging hematology analyzer. As seen from Fig. 2, there is substantial identify as between the simulated cellular components and the cellular components of Fig, 1. In accordancewith the teachings in general, the suspensionmediumof the present teachings is particularly attractive for us with simulated blood cell components (e.g., at least simulated leukocytes, such as stabilized human leukocytes).

[0073] Figs. 3a and 3b are included for comparison.

[0074] Fig. 3a is an enlarged micrograph illustrating respective examples of simulated leukocyte subpopulation cells and other simulated cell components in a suspension composition of the present teachings, the components and suspension composition being freshly prepared, as viewed by optical microscopy (oil immersion lens at 100x), in a form as would be detectable by a digital imaging hematology analyzer.

[0075] For comparison with Fig. 3a, Fig. 3b is an enlarged micrograph illustrating respective examples of leukocyte subpopulation cells and other simulated cell components after storage at about 2 to about 10°C for 105 days in a suspensioncompositionof thepresent teachingsasviewedbyopticalmicroscopy (oil immersion lensat 100x), in a formas would be detectable by a digital imaging hematology analyzer. The image illustrates that as between the freshly prepared materials of Fig. 3a and those of Fig. 3b, there are indistinguishable differences if any. As expected in general for the teachings herein, the nucleic subject matter within the granulated cells, in particular, remains intact through a useful life orders of magnitude longer than it would if not suspending in the suspension medium of the teachings (e.g., at least 105 days).

[0076] Figs. 4a and 4b are included for comparison.

[0077] Fig. 4a is a micrograph to illustrate an example of simulated leukocyte cells in a suspension medium including a stabilizing agent (e.g., a polysaccharide, such as chitosan or another glucosamine, having a polymerization degree ranging from greater than one to about 100) of the present teachings after about three weeks as viewed by optical microscopy (oil immersion lens at 100x), in a form as would be detectable by a digital imaging hematology analyzer. Consistent with the illustrations of the teachings in Figs. 2, 3a and 3b, the cellular components resemble those of fresh whole blood (e.g., fresh human whole blood), as seen in Fig. 1.

[0078] Fig. 4b is a micrograph to illustrate an example of simulated leukocyte cells of the present teachings in a suspension medium as in Fig. 4a, but absent any stabilizing agent of Fig. 4a after about three weeks as viewed by optical microscopy (oil immersion lensat 100x), in a formaswouldbedetectable byadigital imaginghematology analyzer. As can be seen, there is substantial visible degradation. For example, at least the simulated monocytes show considerable transformation from their expected state when suspended in the suspension medium of the present teachings.

[0079] Fig. 5 is amicrograph to illustrate anexample of a prior art hematology control, absent any stabilizing agent of the present teachings, as viewedbyopticalmicroscopy (oil immersion lensat 100x), in a formaswouldbeanalyzedbyadigital imaging hematology analyzer. It can be seen hownuclearmatter is not identifiable. The present application further relates to the following items: 1) A suspension composition adapted for use in assuring quality control of a digital imaging hematology analyzer, comprising: a suspensionmedium(optionally includingabufferedaqueoussolution)withwhichat least onesimulated nucleated blood component is mixed in a predetermined amount, the suspension medium including at least one stabilizing agent in an amount capable of preserving relevant detectable size and morphology, including detectable nuclear morphological characteristics of the nucleated blood component (including any native nuclear cytoplasm granules), when stored at about 2 to about 10°C for a period of at least 3 days from when the at least one simulated nucleated blood component is initially mixed with the suspension medium. 2) A suspension composition for simulated white blood cells of a hematology analyzer control formulation and retaining detectable morphological characteristics of the simulated blood cells, comprising: a. a buffered aqueous solution; and b. at least one stabilizing agent that includes at least one polysaccharide having a polymerization degree ranging from greater than one to about 100, the at least one stabilizing agent being present in an amount sufficient for preserving stability of the relevant detectable characteristics of the simulated blood cells for a period of at least 3 days, after being stored during such period at about 2 to about 10°C, upon suspending the simulated blood cells; wherein upon mixing the suspension composition with the simulated blood cells, the resulting mixture is capable of 10 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 dispensing through a nozzle for delivery to a substrate for analysis by a digital imaging hematology analyzer. 3)Thesuspensioncompositionof item1or2,wherein thesuspensioncomposition isadapted foruse indigital imaging hematology instrument that creates and analyzes an image by a computer implemented technique of a sample that has been dispensed onto a substrate. 4) The suspension composition of any of the above items, wherein the suspension composition is adapted, upon mixingwith the simulatedbloodcells, for dispensing throughanozzle for delivery byprinting to a transparent substrate and subsequent analysis by a digital imaging hematology analyzer, without any material damage (e.g., damage to excess of five percent (5%) by number of total simulated blood cells) to the blood cells. 5) The suspension composition of any of the above items, wherein the at least one stabilizing agent is present in an amount sufficient for preserving stability of the detectable morphological characteristics of the simulated blood cells for a period of at least 30 days, after being stored during such period at about 2 to about 10°C, upon suspending the bloodcells;wherein thesimulatedbloodcells includenucleatedbloodcells, andwherein thedetectablemorphological characteristics that are preserved include size, a nucleus morphology and morphology of other native nuclear cytoplasm granules. 6) The suspension composition of any of the above items,wherein theaqueous buffered solution includes at least one buffering agent, and at least one antimicrobial. 7) The suspension composition of any of the above items,wherein theaqueous buffered solution includes at least one dispersion agent for reducing aggregation of the simulated blood cells as compared with the aqueous buffered solution without the dispersion agent. 8) The suspension composition of any of the above items,wherein the suspension composition has apH ranging from about 6 to about 8. 9) The suspension composition of any of the above items,wherein the at least one stabilizing agent includes least one amino polysaccharide having a polymerization degree ranging from greater than one to about 100. 10) The suspension composition of any of the above items, wherein the at least one stabilizing agent includes least one polysaccharide having a polymerization degree ranging from greater than about 5 to about 40. 11) The suspension composition of any of the above items wherein the at least one stabilizing agent is an oligosaccharide having a weight average molecular weight (measured by high performance liquid chromatography and gel permeation chromatography (HPLC / GPC)) from about 500 to about 5000 (e.g., about 1000 to about 3000). 12) The suspension composition of any of the above itemswherein the at least one stabilizing agent is a glucosamine oligosaccharide having a weight average molecular weight (measured by high performance liquid chromatography and gel permeation chromatography (HPLC / GPC)) from about 1000 to about 3000. 13) The suspension composition of any of the above items, wherein the at least one stabilizing agent includes a glucosamine. 14) The suspension composition of any of the above items, wherein the at least one stabilizing agent includes one or any combination of chitin, a derivative of chitin, a chitosan, a salt of a chitosan or a derivative of a chitosan. 15) The suspension composition of any of the above items wherein the at least one stabilizing agent is selected from D‑(+)‑glucosamine, N-acetyl-D-glucosamine, chitosan acetate, chitosan lactate, chitosan oligosaccharide, carboxyl methyl chitosan, or any combination thereof. 16) The suspension composition of any of the above items, wherein the at least one stabilizing agent is present in an amount up to about ten percent (10%) of the suspension composition. 17) The suspension composition of any of the above items, wherein the simulated blood cells include simulated nucleated leukocytes, nucleated red blood cells or both. 18) The suspension composition of any of the above items, wherein the at least one stabilizing agent is present in an amount up to about ten percent (10%) of a resulting control composition admixture including the suspension composition and the simulated blood cells. 19) The suspension composition of any of the above items, wherein the suspension composition and any resulting control composition employing the suspension composition is substantially free of any added lipoprotein. 20) A white blood cell control composition adapted for use in a digital imaging hematology analyzer comprising the suspension composition of any of the above items. 21) A method of using the suspension composition of any of the above items, comprising a step of dispensing the suspension composition onto a substrate, and at least partially evaporating water from the aqueous buffered suspension composition. EXAMPLES

[0080] Other benefits and advantages of the teachingswill be understood upon reviewof the followingExamples,which provide additional illustrative details of the present teachings. 11 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 Example 1

[0081] ThesuspensioncompositionofTable1 ispreparedandpredeterminedamountsof simulatedbloodcells is added into it. Water makes up the balance. Concentrations are expressed in weight / volume percentages. To the suspension composition, simulated blood cell components are added according to three different predetermined amounts for each to form a resulting control composition. The three amounts correspond respectively to three different levels - a low abnormal level ("L.1"), a normal healthy level ("L.2") and a high abnormal level ("L.3). A stability study is performed. The amounts for each leukocyte sub-population reported are provided in numbers (#) and percentage of the overall leukocyte population (%). Measurements are taken at the time the resulting control composition, day 56 of the study and day 105 of the study. The data are collected on a Roche Cobas m511 hematology Analyzer. After preparation of the resulting control composition, and throughout the duration of the study, the resulting control composition is maintained at a temperature of from about 2 to about 10°C. The results show that the suspensionmedium contributes to stability of the simulated blood cell components of the resulting control composition. This is consistent with the comparative images of Figs. 3a and 3b. Table 1 Ingredient Concentration (Pre-cell Addition)(w / v%) EDTA, Disodium 1.10% Magnesium Chloride 0.03% HEPES 1.19% Calcium Acetate 0.05% Polyethylene Glycol 20,000 1.00% Urea 0.50% Sodium Chloride 2.64% Chloramphenicol 0.01% Sodium Azide 0.10% Bovine Serum Albumin 1.00% Chitosan Oligosaccharide 0.50% Sodium Hydroxide 0.18%

[0082] The composition is analyzed using a Roche Cobas m511™ analyzer, with all levels analyzed in triplicate and reported as theaverage of the three analyses. Tests are performedon two lots of compositions. Results are reported in the below Tables 2 and 3, respectively for the first lot and the second lot. 12 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 13 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55

[0083] For the above tables the following abbreviations are used: WBC: white blood cell; RBC: red blood cell; HGB: hemoglobin;HCT:hematocrit;MCV:meancorpuscular volume;MCH:meancorpuscularmass;MCHC:meancorpuscular hemoglobin concentration; PLT: platelet; RDW: redblood cell distributionwidth; SDRDW: standard deviation for red blood cell distributionwidth;MPV:meanplatelet volume; nRBC:nucleated redbloodcell;RETIC: reticulocyte;NEUT:neutrophil; LYM: lymphocyte; MONO: monocyte; EOS: eosinophil; BASO: basophil. General Remarks Applicable to All Teachings

[0084] As used herein, unless otherwise stated, the teachings envision that any member of a genus (list) may be excluded from the genus; and / or any member of a Markush grouping may be excluded from the grouping.

[0085] Unless otherwise stated, any numerical values recited herein include all values from the lower value to the upper value in incrementsof oneunit provided that there isa separationofat least 2units betweenany lower valueandanyhigher value. As an example, if it is stated that the amount of a component, a property, or a value of a process variable such as, for example, temperature, pressure, time and the like is, for example, from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70, it is intended that intermediate range values such as (for example, 15 to 85, 22 to 68, 43 to 51 , 30 to 32 etc.) arewithin the teachingsof this specification.Likewise, individual intermediate valuesarealsowithin thepresent teachings. For values which are less than one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similarmanner. As can be seen, the comparative teaching of amounts expressed as weight / volume percent for two or more ingredients also encompasses relative weight proportions of the two or more ingredients to each other, even if not expressly stated. For example, if a teaching recites 2% A, and 5% B, then the teaching also encompasses a weight ratio of A:B of 2:5. Unless otherwise stated, all ranges include both endpoints and all numbers between the endpoints. The use of "about" or "approximately" in connection with a range applies to both ends of the range. Thus, "about 20 to 30" is intended to cover "about 20 to about 30", inclusive of at least the specified endpoints.

[0086] Thedisclosures of all articles and references, including patent applications andpublications, are incorporated by reference for all purposes.

[0087] The term "consisting essentially of to describe a combination shall include the elements, ingredients, compo- nents or steps identified, and such other elements ingredients, components or steps that do notmaterially affect the basic and novel characteristics of the combination. The use of the terms "comprising" or "including" to describe combinations of 14 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 elements, ingredients, components or steps herein also contemplates embodiments that consist essentially of (namely, thepresenceofanyadditional elements, ingredients, componentsorsteps, doesnotmaterially affect thepropertiesand / or benefits derived from the teachings; or even consist of the elements, ingredients, components or steps.

[0088] Plural elements, ingredients, components or steps can be provided by a single integrated element, ingredient, component or step. Alternatively, a single integrated element, ingredient, component or step might be divided into separate plural elements, ingredients, components or steps. The disclosure of "a" or "one" to describe an element, ingredient, component or step is not intended to foreclose additional elements, ingredients, components or steps. All references herein to elements ormetals belonging to a certainGroup refer to thePeriodic Table of theElements published and copyrighted by CRC Press, Inc., 1989. Any reference to the Group or Groups shall be to the Group or Groups as reflected in this Periodic

[0089] Table of the Elements using the IUPAC system for numbering groups.

[0090] It is understood that the above description is intended to be illustrative and not restrictive.Many embodiments as well asmanyapplications besides theexamplesprovidedwill beapparent to thoseof skill in theart upon reading theabove description. The scope of the teachings should, therefore, be determined not with reference to the above description, but should insteadbedeterminedwith reference to theappendedclaims, alongwith the full scopeof equivalents towhich such claims are entitled. The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes. The omission in the following claims of any aspect of subject matter that is disclosed herein is not a disclaimer of such subject matter, nor should it be regarded that the inventors did not consider such subject matter to be part of the disclosed inventive subject matter.

[0091] The explanations and illustrations presented herein are intended to acquaint others skilled in the art with the teachings, its principles, and its practical application. Those skilled in the art may adapt and apply the teachings in its numerous forms, as may be best suited to the requirements of a particular use. Accordingly, the specific embodiments of the present teachings as set forth are not intended as being exhaustive or limiting of the teachings. The scope of the teachings should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes. Other combinations are also possible as will be gleaned from the following claims, which are also hereby incorporated by reference into this written description. Claims 1. A suspension composition comprising: one or more simulated blood components, wherein the one ore more simulated blood components are derived from one or more processed blood cells; a buffered aqueous solution; and at least onestabilizing agent,wherein theat least one stabilizingagent includesa chitosanand / or chitin, a salt of a chitosan and / or chitin, and / or some other derivative of a chitosan and / or chitin. 2. The suspension composition of claim 1, wherein the suspension composition is adapted for use in digital imaging hematology instrument that creates and analyzes an image by a computer implemented technique of a sample that has been dispensed onto a substrate. 3. The suspension composition of claim 1 or 2, wherein the suspension composition is adapted, upon mixing with the simulated blood cells, for dispensing through a nozzle for delivery by printing to a transparent substrate and subsequent analysis by a digital imaging hematology analyzer. 4. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is present in an amount sufficient for preserving stability of the detectable morphological characteristics of the simulated blood cells for a period of at least 30 days, after being stored during such period at about 2 to about 10°C, upon suspending the blood cells; wherein the simulated blood cells include nucleated blood cells, and wherein the detectable morphological characteristics that are preserved include size, a nucleus morphology and morphology of other native nuclear cytoplasm granules. 5. The suspension composition of any of the above claims, wherein the aqueous buffered solution includes at least one 15 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 buffering agent, and at least one antimicrobial. 6. The suspension composition of any of the above claims, wherein the aqueous buffered solution includes at least one dispersion agent for reducing aggregation of the simulated blood cells as compared with the aqueous buffered solution without the dispersion agent. 7. The suspension composition of any of the above claims, wherein the suspension composition has a pH ranging from about 6 to about 8. 8. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is selected from chitosan acetate, chitosan lactate, chitosan oligosaccharide, carboxyl methyl chitosan, or any combination thereof. 9. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is present in an amount up to about ten percent (10%) or about seven percent (7%) or about five percent (5%) of the suspension composition. 10. The suspension composition of any of the above claims, wherein the simulated blood cells include simulated nucleated leukocytes, nucleated red blood cells or both. 11. The suspension composition of claim 10, wherein the simulated nucleated leukocytes include a three-part leukocyte populationofwholebloodof neutrophils, lymphocytesandmonocytes, orwherein thesimulatednucleated leukocytes include a five-part leukocyte population of whole blood of neutrophils, eosinophils, basophils, lymphocytes and monocytes. 12. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is present in an amount up to about ten percent (10%) of a resulting control composition admixture including the suspension composition and the simulated blood cells. 13. The suspension composition of any of the above claims, wherein the suspension composition and any resulting control composition employing the suspension composition is substantially free of any added lipoprotein. 14. The suspension composition of any of the above claims, wherein the simulated blood cells include a simulated platelet, reticulated platelet, red blood cell, reticulocyte, immature reticulocyte, nucleated red blood cell, or any combination thereof. 15. Thesuspension compositionof anyof theaboveclaims,wherein theamounts of theoneormore simulatedblood cells is a known amount for simulating normal amounts in whole blood. 16. A method of using the suspension composition of any of the above claims, comprising a step of dispensing the suspension composition onto a substrate, and at least partially evaporating water from the aqueous buffered suspension composition. 17. Themethod of claim 16, further comprisingmaking and analyzing an image of the suspension composition in a digital imaging haematology instrument. 16 EP 4 656 715 A2 5 10 15 20 25 30 35 40 45 50 55 17 EP 4 656 715 A2 18 EP 4 656 715 A2 19 EP 4 656 715 A2 20 EP 4 656 715 A2 21 EP 4 656 715 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description • US 4160644 A

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[0034] • US 5250438 A

[0062] Non-patent literature cited in the description • Periodic Table of theElements. CRCPress, Inc, 1989

[0088] (19) *EP004656715A3* (11) EP 4 656 715 A3 (12) EUROPEAN PATENT APPLICATION (88) Date of publication A3: 25.02.2026 Bulletin 2026 / 09 (43) Date of publication A2: 03.12.2025 Bulletin 2025 / 49 (21) Application number: 25204066.2 (22) Date of filing: 28.07.2017 (51) International Patent Classification (IPC): G01N 33 / 50 (2006.01) C12N 5 / 078 (2010.01) G01N 33 / 569 (2006.01) G01N 33 / 96 (2006.01) (52) Cooperative Patent Classification (CPC): G01N 33 / 5094; C12N 5 / 0634; G01N 33 / 56972; G01N 33 / 96 (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 29.07.2016 US 201662368676 P 03.02.2017 US 201762454224 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 17757593.3 / 3 491 381 (71) Applicant: Streck LLC La Vista, NE 68128 (US) (72) Inventors: • HUNSLEY, Brad Papillion, NE 68133 (US) • SCHOLL, John Omaha, NE 68135 (US) (74) Representative: Stolmár & Partner Patentanwälte PartG mbB Blumenstraße 17 80331 München (DE) (54) SUSPENSION COMPOSITION FOR HEMATOLOGY ANALYSIS CONTROL (57) A suspension composition for a hematology analysis control particularly useful for preserving relevant detectable characteristics of blood cells for a prolong stability period. The suspension may include at least one polysaccharide, which may include or derive from chitosan and / or chitin, as a stabilizing agent. EP 4 65 6 71 5 A 3 Processed by Luminess, 75001 PARIS (FR) 2 EP 4 656 715 A3 5 10 15 20 25 30 35 40 45 50 55 3 EP 4 656 715 A3 5 10 15 20 25 30 35 40 45 50 55 {SCHPY-26038-HKSPT / 02506707v1} 摘要 一种用于血液学分析质控品的悬浮液组合物，特别适用于延长血液细胞相关可检测特 征的稳定保存期。该悬浮液可包含至少一种多糖作为稳定剂，所述多糖可包括或源自 壳聚糖和 / 或几丁质。

Claims

1. A suspension composition comprising: one or more simulated blood components, wherein the one ore more simulated blood components are derived from one or more processed blood cells; a buffered aqueous solution; and at least one stabilizing agent, wherein the at least one stabilizing agent includes a chitosan and / or chitin, a salt of a chitosan and / or chitin, and / or some other derivative of a chitosan and / or chitin.

2. The suspension composition of claim 1, wherein the suspension composition is adapted for use in digital imaging hematology instrument that creates and analyzes an image by a computer implemented technique of a sample that has been dispensed onto a substrate.

3. The suspension composition of claim 1 or 2, wherein the suspension composition is adapted, upon mixing with the simulated blood cells, for dispensing through a nozzle for delivery by printing to a transparent substrate and subsequent analysis by a digital imaging hematology analyzer.

4. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is present in an amount sufficient for preserving stability of the detectable morphological characteristics of the simulated blood cells for a period of at least 30 days, after being stored during such period at about 2 to about 10°C, upon suspending the blood cells; wherein the simulated blood cells include nucleated blood cells, and wherein the detectable morphological characteristics that are preserved include size, a nucleus morphology and morphology of other native nuclear cytoplasm granules.

5. The suspension composition of any of the above claims, wherein the aqueous buffered solution includes at least one buffering agent, and at least one antimicrobial.

6. The suspension composition of any of the above claims, wherein the aqueous buffered solution includes at least one dispersion agent for reducing aggregation of the simulated blood cells as compared with the aqueous buffered solution without the dispersion agent.

7. The suspension composition of any of the above claims, wherein the suspension composition has a pH ranging from about 6 to about 8.

8. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is selected from chitosan acetate, chitosan lactate, chitosan oligosaccharide, carboxyl methyl chitosan, or any combination thereof.

9. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is present in an amount up to about ten percent (10%) or about seven percent (7%) or about five percent (5%) of the suspension composition.

10. The suspension composition of any of the above claims, wherein the simulated blood cells include simulated nucleated leukocytes, nucleated red blood cells or both.

11. The suspension composition of claim 10, wherein the simulated nucleated leukocytes include a three-part leukocyte population of whole blood of neutrophils, lymphocytes and monocytes, or wherein the simulated nucleated leukocytes include a five-part leukocyte population of whole blood of neutrophils, eosinophils, basophils, lymphocytes and monocytes.

12. The suspension composition of any of the above claims, wherein the at least one stabilizing agent is present in an amount up to about ten percent (10%) of a resulting control composition admixture including the suspension composition and the simulated blood cells.

13. The suspension composition of any of the above claims, wherein the suspension composition and any resulting control composition employing the suspension composition is substantially free of any added lipoprotein.

14. The suspension composition of any of the above claims, wherein the simulated blood cells include a simulated platelet, reticulated platelet, red blood cell, reticulocyte, immature reticulocyte, nucleated red blood cell, or any combination thereof.

15. The suspension composition of any of the above claims, wherein the amounts of the one or more simulated blood cells is a known amount for simulating normal amounts in whole blood.

16. A method of using the suspension composition of any of the above claims, comprising a step of dispensing the suspension composition onto a substrate, and at least partially evaporating water from the aqueous buffered suspension composition.

17. The method of claim 16, further comprising making and analyzing an image of the suspension composition in a digital imaging haematology instrument.

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