WO2019090126A1

WO2019090126A1 - Linearity control materials

Info

Publication number: WO2019090126A1
Application number: PCT/US2018/059028
Authority: WO
Inventors: Bradford A. HUNSLEY; John W. SCHOLL; Kitty MCCARTHY; Cathy HORSTMAN
Original assignee: Streck, Inc.
Priority date: 2017-11-03
Filing date: 2018-11-02
Publication date: 2019-05-09

Abstract

A control for resembling a predetermined concentration of an erythrocyte population, comprising at least one first cellular component including a plurality of simulated red blood cells, wherein the at least one first cellular component composed of erythrocytes that have been treated or altered from their naturally occurring state and upon passing through an automated hematology analyzer that analyzes the cellular components by a digital imaging technique are detected as erythrocytes and in an amount corresponding to a known predetermined amount.

Description

LINEARITY CONTROL MATERIALS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No,

62/581 ,643, filed November 3, 2017, the disclosure of which is incorporated by reference in its entirety,

FIELD

[0002] The present invention relates to a linearity reference control composition and a linearity control system that enables verification of the reportable measurement range and linearity of the measurements of hematology analyzers utilizing digital imaging for one or more of white blood cells, red blood cells and other cellular components.

BACKGROUND OF THE INVENTION

[0003] Quality control in clinical hematology is critical for accurate testing of patient samples. Such accuracy in the counting of blood ceils is dependent upon the use of adequate control products. Historically, maintaining a quality control program for blood ceil counting involved the use of fresh human blood as a whole blood standard. However, fresh blood is usable for a limited period of time, requiring the development of man factured simulated control products which have longer life.

[0004] Various commercial hematology analyzers are available and the way in which these devices analyze the blood samples often differs from device to device. As a result, a variety of simulated blood control compositions are available commercially to operate with the wide range of analyzers. Additional controls, commonly referred to as linearity controls, are also required for testing the reportable range of various component measurements on hematology analyzers and determine linearity of the reported parameters. Such linearity controls typically contain a single blood ceil component at various concentrations over reportable range of a specific instrument.

[0005] U.S. Pat. No. 9,459,196 (incorporated by reference herein for all purposes) teaches a system for displaying performance data generated from blood analyzers and hematology control for use with the system. U.S. Patent Publication No. 2009/0238438 (incorporated by reference herein for ail purposes) teaches a hematology analyzer using optical density measurements to determine hemoglobin concentration. U.S. Patent No. 8,488,1 1 1 (incorporated by reference herein for all purposes) teaches a hematology analyzer that utilizes digital images to determine optical density and the associated mean cell volume of blood components. [0006] The analyzers that utilize digital images require quality control compositions that contain simulated cells that will provide the necessary visual consistency to appear as the desired cell components. Whereas most simulated controls must only reflect one particular measurement that matches a blood component of interest, digital image controls must actually look like the blood component of interest. As an example, the color or relative color of a control blood component must match that of an actual whole blood component. Consequently, there is a strong need for improved linearity control compositions and an improved linearity control system that can be utilized in a digital imaging analyzer.

SUMMARY OF THE INVENTION

[0007] Accordingly, in some aspects the disclosure provides a method of making a control for resembling a predetermined concentration of an erythrocyte, reticulocyte, nucleated red blood ceil, leukocyte or thrombocyte population, comprising the steps of admixing in a diluent: at least one cellular component comprising a plurality of cells , such that upon passing through an automated hematology analyzer that analyzes the cellular component by a digital imaging technique, the ceils are detected as erythrocytes, reticulocytes, nucleated red blood cells, leukocytes or thrombocytes, and in a concentration corresponding to a known predetermined concentration, in some aspects, the disclosure provides a method of making a control for resembling a predetermined concentration of cell population, comprising the steps of admixing in a diluent: at least one cellular component comprising a plurality of cells , such that upon passing through an automated hematology analyzer that analyzes the cellular component by a digital imaging technique, the cells are detected as erythrocytes, reticulocytes, nucleated red blood ceils, leukocytes or thrombocytes, and in a concentration corresponding to a known predetermined concentration. In some embodiments, the ceils comprise a red blood ceil analog, a reticulocyte analog, a nucleated red blood cell analog, a white blood cell analog, a platelet analog, or a combination thereof, in further embodiments, the simulated red blood ceil comprises a processed human erythrocyte, an animal erythrocyte, a particuiated synthetic material or a combination thereof.

[0008] In some embodiments, the admixing is performed at or above room temperature, in further embodiments, the diluent comprises water, alcohol, or a combination thereof,

[0009] In some embodiments, the method includes a step of providing the plurality of cells from a first source and having a first color corresponding with a first range of grayscale values and treating the plurality of ceils to alter the color to a second color corresponding to a second range of grayscale values. In still further embodiments, the step of treating includes subjecting the cells to a fluid that includes at least about 25% by volume carbon monoxide, alcohol, or a combination thereof. In some embodiments, the alcohol is selected from a C1 to C6 alcohol, a dioi, a triol, or a combination thereof,

[0010] In any of the embodiments of the disclosure, a method as described herein further includes treating the at least one cellular component with a preservative agent. In some embodiments, the preservative agent includes glyceraidehyde, DL benzisothiazolinone, phthaldiaidehyde, diazolidinyl urea, Imidazolidinyi urea, formaldehyde, formalin, glutaraidehyde, or a combination thereof,

[0011] in any of the embodiments of the disclosure, a method as described herein further includes treating the at least one cellular component for reducing rouleaux, reducing aggregation, reducing cell stickiness, or a combination thereof.

[0012] In further embodiments, the at least one cellular component is contacted with a phosphate buffered saline (PBS).

[0013] In any of the embodiments of the disclosure, a method as described herein further includes a plurality of sub-treatments separated by at least one washing step for removing a reagent from a previous step, in any of the embodiments of the disclosure, a method as described herein further includes exposing the control to illumination at a plurality of different wavelengths. In some embodiments, the wavelengths of the illumination are from about 300 nm to about 900 nm. In further embodiments, the wavelengths of the illumination are from about 400 nm to about 650 nm. in some embodiments, the at least one cellular component is stainable to provide for optical detection. In still further embodiments, the at least one ceiiular component undergoes automated measurement of at least one optical density value.

[0014] in some aspects, the disclosure provides a cellular fraction for an erythrocyte, reticulocyte, nucleated red blood cell, leukocyte, or thrombocyte linearity control, comprising: a predetermined concentration of at least one cellular component including a piurality of cells, such that upon passing through an automated hematology analyzer that analyzes the cellular components by a digital imaging technique, the cells are detected as erythrocytes, reticulocytes, nucleated red blood cells, leukocytes, or thrombocytes, and in a concentration corresponding to a known predetermined concentration, in further aspects, the disclosure provides a cellular fraction for a ceiiular linearity control, comprising: a predetermined concentration of at least one cellular component including a plurality of cells, such that upon passing through an automated hematology analyzer that analyzes the cellular components by a digital imaging technique, the cells are detected as erythrocytes, reticulocytes, nucleated red blood cells, leukocytes, or thrombocytes, and in a concentration corresponding to a known predetermined concentration. [0015] in some embodiments, the cells comprise a red blood cell analog, a reticulocyte analog, a nucleated red blood cell analog, a white blood cell analog, a platelet analog, or a combination thereof, in further embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 0 to about 245 for 8-bit grayscale. In some embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 50 to about 200 for 8-bit grayscale, in further embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 0 to about 100 for 8-bit grayscale, in still further embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of 100 to 240 for 8-bit grayscale. In some embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 20 to about 80 for 8-bit grayscale. In some embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of at least about 20 for 8-bit grayscale. In some embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of at least about 50 for 8-bit grayscale. In further embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of less than about 150 for 8-bit grayscale, in still further embodiments, the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of less than about 100 for 8-bit grayscale.

[0016] in some embodiments, the visual color difference in grayscale between the cells and a non-erythrocyte is from about 0.5 to 13.5 according to test method ASTM D2616-12. in further embodiments, the visual color difference in grayscale between the ceils and a non- erythrocyte is from about 1 to 10 according to test method ASTM D2616-12. in still further embodiments, the visual color difference in grayscale between the ceils and a non-erythrocyte is from about 6 to 12 according to test method ASTM D2616-12.

[0017] In some embodiments, the cells resemble erythrocytes from a human blood sample in one or more of size and morphology. In some embodiments, the cells have a mean corpuscular volume of from about 30 to about 150 f L, or even from about 80 f L to about 100 fL. In further embodiments, the cells have a mean corpuscular volume of from about 20 to about 70 fL In still further embodiments, the ceils have a mean corpuscular volume of from about 1 10 to about 150 fL. In some embodiments, the cells are preserved.

[0018] In some embodiments, the red blood ceil analog comprises a processed human erythrocyte, an animal erythrocyte, a participated synthetic material or a combination thereof.

[0019] in some embodiments, the cellular fraction further includes at least one additional cellular component wherein upon passing through an automated hematology analyzer cells of the at least one second additional component are detected by the analyzer as a white blood cell. In some embodiments, the at least one cellular component has a size prior to processing from about 80 fL to about 100 fL, or even about 150 fL to about 200 fL. in some embodiments, the erythrocytes, reticulocytes, nucleated red blood cells, leukocytes, or thrombocytes are fixed and/or swollen by contact with a solution containing giutaraidehyde, dimethyl sulfoxide, giyceraldehyde, DL benzisothiazolinone, phthaidialdehyde, diazolidinyl urea, imidazolidinyl urea, formaldehyde, formalin, or any combination thereof. In some embodiments, the ceils are alligator blood cells. In some embodiments, the cellular fraction further includes an erythrocyte contacted by a surfactant selected from poioxamers, polyethylene glycol, octylpheno! ethoxylate, Polyoxyethanyl a-tocopheryl sebacate (PTS), DL-a-tocopherol methoxypolyethylene glycol succinate, or a combination thereof.

[0020] in some embodiments, the at least one cellular component is exposed to an elevated temperature of greater than 30 ^QC, greater than 50 ^QC, or greater than 70 ^eC. In further embodiments, the at least one cellular component includes a membrane that has been cross- linked or otherwise denatured, in still further embodiments, the at least one cellular component comprises a processed red blood ceil derived from alligators, cattle, porcine, equine, newts, salamanders, turkeys, geese, chickens, sharks, cows, pigs, goats, salmon, human, or trout. In some embodiments, the at least one cellular component includes a nucleus. In further embodiments, the at least one cellular component is free of any nucleus.

[0021] in some embodiments, the at least one cellular component is preserved and then washed after preservation. In some embodiments, a method of the disclosure further includes a step of treating the at least one cellular component so that the cellular component appears red in color. In further embodiments, the at least one cellular component is contacted with methylene blue. In some embodiments, a method of the disclosure further includes one or more shrinking or swelling steps. [0022] in some aspects, the disclosure provides a cellular fraction formed by any of the methods of the disclosure, in further aspects, the disclosure provides a linearity control made with a cellular fraction of the disclosure.

[0023] in some aspects, the disclosure provides a method of operating an optical imaging hematology analyzer comprising running a linearity control made using a cellular fraction of the disclosure on the analyzer, in some embodiments, the at least one cellular component is suspended in a matrix material and the matrix material has a viscosity of from about 0.01 to about 5,0 cP. In further embodiments, a method of the disclosure further includes a step of diluting the control.

[0024] in any of the embodiments of the disclosure, the diluent is selected from water, para 12, para 8, elhySenediaminetetraacetic acid (EDTA), magnesium gluconate, polyethylene glycol, soybean based protease inhibitor, and combinations thereof. In further embodiments, the control includes one or more of antipain, aprotinin, chymostatin, eiastatinal, phenylmethylsuifonyl fluoride (PMSF), APMSF, TICK, TPCK, ieupeptin, soybean trypsin inhibitor, indoieacetic acid (!AA), E-64, pepstatin, VdLPFFVdL, EDTA, 1 ,10-phenanthro!ine, phosphoramodon, amastatin, bestatin, diprotin A, diprotin B, aipha-2-macrogiobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin egg white cystatin, and any combination thereof.

[0025] in any of the embodiments of the disclosure, protective agent includes a phosphatase inhibitor selected from the group consisting of: ca!ycu!in A, noduiarin, NiPP-1 , microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin, endothall, noduiarin, cyclosporin A, FK 506/immunophiiin complexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic) DMHV, sodium orthovanadate, and a combination thereof.

[0026] in one aspect, the present invention is directed to a method of making a control for resembling a predetermined concentration of an erythrocyte population, comprising the steps of admixing in a diluent: at least one first cellular component including a plurality of simulated red blood cells; wherein the at least one first cellular component is composed of erythrocytes that have been treated or altered from their naturally occurring state and upon passing through an automated hematology analyzer that analyzes the cellular components by a digital imaging technique are detected as erythrocytes and in an amount corresponding to a known predetermined amount.

[0027] The simulated red blood cells may comprise processed human erythrocytes, animal erythrocytes, a particulated synthetic material or a combination thereof. The admixing may be performed at or above room temperature (ambient) and/or with a diluent comprising water, alcohol, or both. The method may include a step of providing a plurality of erythrocytes from a first source and having a first color corresponding with a first range of grayscale values and treating the plurality of erythrocytes to alter the color to a second color corresponding to a second range of grayscale values. The step of treating may include subjecting the erythrocytes to a fluid that includes at least about 25% by volume carbon monoxide, alcohol, or a combination thereof. The alcohol may be selected from a C1 to C6 alcohol, a diol, a triol, or any combination thereof.

[0028] The method may include treating the at least one first cellular component with a preservative agent. The preservative agent may include glyceraldehyde, benzisothiazolinone, phthaldialdehyde, diazolidinyl urea, Imidazolidinyl urea, formaldehyde, formalin, glutaraldehyde, or any combination thereof. The method may include treating the at least one first cellular component for reducing rouleaux, reducing aggregation, reducing cell stickiness, or any combination thereof. The at least one first cellular component may be contacted with a phosphate buffered saline (PBS). The method may include a plurality of sub-treatments separated by at least one washing step for removing a reagent from a previous step.

[0029] The method may include exposing the control to illumination at a plurality of different wavelengths. The wavelengths of the illumination may be from about 300 nm to about 900 nm. The wavelengths of the illumination may be from about 400 nm to about 650 nm. The at least one first cellular component may be stainable to provide for optical detection. The at least one first cellular component may undergo automated measurement of at least one optical density value.

[0030] The teachings herein may further provide for a cellular fraction for an erythrocyte linearity control, comprising: a predetermined concentration of at least one first cellular component including a plurality of simulated red blood cells; wherein the at least one first cellular component is composed of erythrocytes that have been treated or altered from their naturally occurring state and upon passing through an automated hematology analyzer that analyzes the cellular components by a digital imaging technique are detected as erythrocytes and in an amount corresponding to a known predetermined amount.

[0031] The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 0 to about 245 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 50 to about 200 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 0 to about 100 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of 100 to 240 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 20 to about 80 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of at least about 20 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of at least about 50 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of less than about 150 for 8-bit grayscale. The erythrocytes when imaged using a focal plane array may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of less than about 100 for 8-bit grayscale.

[0032] The visual color difference in grayscale between the erythrocytes and a non- erythrocyte may be from about 0.5 to 13.5 according to test method ASTM D2616-12. The visual color difference in grayscale between the erythrocytes and a non-erythrocyte may be from about 1 to 10 according to test method ASTM D2616-12. The visual color difference in grayscale between the erythrocytes and a non-erythrocyte may be from about 6 to 12 according to test method ASTM D2616-12.

[0033] The erythrocytes may resemble erythrocytes from a human blood sample in one or more of size and morphology. The erythrocytes may have a mean corpuscular volume of from about 30 to about 150 fL, or even from about 80 fL to about 100 fL. The erythrocytes may have a mean corpuscular volume of from about 20 to about 70 fL. The erythrocytes may have a mean corpuscular volume of from about 1 10 to about 150 fL.

[0034] The erythrocytes may be preserved. The simulated red blood cells may comprise processed human erythrocytes, animal erythrocytes, a particulated synthetic material or a combination thereof. The cellular fraction may include at least one second cellular component wherein upon passing through an automated hematology analyzer cells of the at least one second cellular component are detected by the analyzer as a white blood cell. The at least one first cellular component may have a size prior to processing from about 80 fL to about 100 fL, or even about 150 fL to about 200 fL. [0035] The erythrocytes may be fixed and/or swollen by contact with a solution containing glutaraldehyde, dimethyl sulfoxide, glyceraldehyde, DL benzisothiazolinone, phthaldialdehyde, diazolidinyl urea, Imidazolidinyl urea, formaldehyde, formalin, or any combination thereof. The erythrocytes may be alligator blood cells. The erythrocytes may be contacted by a surfactant selected from poloxamers, polyethylene glycol, octylphenol ethoxyiate, Polyoxyethanyl a-tocopheryl sebacate (PTS), DL-a-tocopherol methoxypolyethylene glycol succinate, or combinations thereof. The at least one cellular component may be exposed to an elevated temperature of greater than 30 ^eC, greater than 50 ^eC, or even greater than 70 ^eC. The at least one first cellular component includes a membrane that has been cross-linked or otherwise denatured. The at least one first cellular component may be a processed red blood cell derived from alligators, cattle, porcine, equine, newts, salamanders, turkeys, geese, chickens, sharks, cows, pigs, goats, salmon, human, trout, or another source of a blood cell. The at least one first cellular component may include a nucleus. The at least one first cellular component may be free of any nucleus.

[0036] In another aspect, the teachings herein provide for a cellular fraction formed by the methods described herein. The teachings herein for provide for a linearity control composition made with the cellular fraction as described herein.

[0037] The at least one cellular fraction may be preserved and then washed after preservation. The methods described herein may include a step of treating the at least one first cell fraction so that the cell fraction appears red in color. The at least one cellular fraction may be contacted with methylene blue. The methods described herein may include one or more shrinking or swelling steps.

[0038] In yet another aspect, the teachings herein are directed to a method of operating an optical imaging hematology analyzer comprising running a linearity control made using the cellular fractions described herein.

[0039] The at least one cellular fraction may be suspended in a matrix material and the matrix material may have a viscosity of from about 0.01 to about 5.0 cP. The method may include a step of diluting the control. The diluent may be selected from water, para 12, para 8, ethylenediaminetetraacetic acid (EDTA), magnesium gluconate, polyethylene glycol, soybean based protease inhibitor, and combinations thereof. The control may include one or more of antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E-64, pepstatin, VdLPFFVdL, EDTA, 1 ,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin egg white cystatin, and any combination thereof. The protective agent may include a phosphatase inhibitor selected from the group consisting of: calyculin A, nodularin, NIPP-1 , microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin, endothall, nodularin, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic) DMHV, sodium orthovanadate, or combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present teachings provide a linearity reference control composition that comprises simulated erythrocytes in a diluent, wherein the stabilized erythrocytes are treated to have a color in grayscale that matches that of an erythrocyte of a fresh blood sample. Optionally, the linearity control composition can further comprise reticulocyte analogs, nucleated red blood cell analogs, white blood cell analogs, platelet analogs, or any combination thereof,

[0041] The linearity control of the teachings herein may be formulated by admixing in a diluent a first cellular component including a plurality of simulated red blood cells. The first cellular component may be composed of erythrocytes that have been treated or altered from their naturally occurring state. Upon passing through an automated hematology analyzer that analyzes the cellular components by a digital imaging technique, the erythrocytes may be detected as erythrocytes and in an amount corresponding to a known predetermined amount. The admixing may be performed at or above room temperature (ambient) and/or with a diluent comprising water, alcohol, or a combination thereof.

[0042] The simulated red blood cells may comprise processed human erythrocytes, animal erythrocytes, a particulated synthetic material or a combination thereof. The at least one first cellular component may be a processed blood cell derived from alligators, cattle, porcine, equine, newts, salamanders, turkeys, geese, chickens, sharks, cows, pigs, goats, salmon, human, trout, or another source of a blood cell. The simulated blood cells may include a nucleus. The simulated blood cells may be free of any nucleus.

[0043] The simulated blood cells may be treated to have a desired color. Such desired color may be a grayscale color. One or more of the cellular components may have a first color corresponding with a first range of grayscale values. The cellular components may then be treated to alter their color to a second color corresponding to a second range of grayscale values. The step of treating may include subjecting the erythrocytes to a fluid that includes at least about 25% by volume carbon monoxide, alcohol, or a combination thereof. The alcohol may be selected from a C1 to C6 alcohol, a diol, a triol, or any combination thereof. [0044] One or more components of the linearity control may be preserved. The preservative agent may include glyceraldehyde, benzisothiazolinone, phthaldialdehyde, diazolidinyl urea, Imidazolidinyl urea, formaldehyde, formalin, glutaraldehyde, or any combination thereof. One or more components of the linearity control may be treated for reducing rouleaux, reducing aggregation, reducing cell stickiness, or any combination thereof. This may be accomplished by contact with a phosphate buffered saline (PBS). One or more components of the linearity control may be subjected to a plurality of sub-treatments separated by at least one washing step for removing a reagent from a previous step.

[0045] When the linearity controls described herein are utilized in a hematology analyzer, they may be exposed to illumination at a plurality of wavelengths. The wavelengths of the illumination may be from about 300 nm to about 900 nm. The wavelengths of the illumination may be from about 400 nm to about 650 nm. One or more blood components may be stainable to provide for optical detection in a hematology analyzer. One or more blood components may undergo automated measurement of at least one optical density value in a hematology analyzer.

[0046] The simulated erythrocytes, when imaged using a focal plane array, may produce a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 0 to about 245 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of from about 50 to about 200 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of from about 0 to about 100 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of 100 to 240 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of from about 20 to about 80 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of at least about 20 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of at least about 50 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of less than about 150 for 8-bit grayscale. The erythrocytes may have a grayscale reading in the range of less than about 100 for 8-bit grayscale.

[0047] The visual color difference in grayscale between the erythrocytes and a non- erythrocyte may be from about 0.5 to 13.5 according to test method ASTM D2616-12. The visual color difference in grayscale between the erythrocytes and a non-erythrocyte may be from about 1 to 10 according to test method ASTM D2616-12. The visual color difference in grayscale between the erythrocytes and a non-erythrocyte may be from about 6 to 12 according to test method ASTM D2616-12. The visual color difference in grayscale between the erythrocytes prior to and posy treatment may be from about 0.5 to 13.5 according to test method ASTM D2616-12. The visual color difference in grayscale between the erythrocytes prior to and post treatment may be from about 1 to 10 according to test method ASTM D2616-12. The visual color difference in grayscale between the erythrocytes prior to and post treatment may be from about 6 to 12 according to test method ASTM D2616-12.

[0048] The simulated erythrocytes after treatment may resemble erythrocytes from a human blood sample in one or more of size and morphology. As a result, the erythrocytes may have a mean corpuscular volume of from about 30 to about 150 fl_, or even from about 80 fl_ to about 100 fl_. The erythrocytes may have a mean corpuscular volume of from about 20 to about 70 fl_. The erythrocytes may have a mean corpuscular volume of from about 1 10 to about 150 fl_. The at least one first cellular component may have a size prior to processing from about 80 f L to about 100 f L, or even about 150 f L to about 200 f L.

[0049] The erythrocytes may be fixed and/or swollen by contact with a solution containing glutaraldehyde, dimethyl sulfoxide, glyceraldehyde, DL benzisothiazolinone, phthaldialdehyde, diazolidinyl urea, Imidazolidinyl urea, formaldehyde, formalin, or any combination thereof. The erythrocytes may be alligator blood cells. The erythrocytes may be contacted by a surfactant selected from poloxamers, polyethylene glycol, octyiphenol ethoxylate, Polyoxyethanyl a-tocopheryl sebacate (PTS), DL-a-tocopherol methoxypolyethylene glycol succinate, or combinations thereof. The at least one cellular component may be exposed to an elevated temperature of greater than 30 ^eC, greater than 50 ^eC, or even greater than 70 ^eC. The at least one first cellular component includes a membrane that has been cross-linked or otherwise denatured.

[0050] The methods described herein may include a step of treating the at least one first cell fraction so that the cell fraction appears red in color. The at least one cellular fraction may be contacted with methylene blue, carbon monoxide and/or alcohol.

[0051] The at least one cellular fraction of the simulated cells may be suspended in a matrix material and the matrix material may have a viscosity of from about 0.01 to about 5.0 cP. The method may include a step of diluting the control. The diluent may be selected from water, para 12, para 8, ethylenediaminetetraacetic acid (EDTA), magnesium gluconate, polyethylene glycol, soybean based protease inhibitor, and combinations thereof. The control may include one or more of antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E- 64, pepstatin, VdLPFFVdL, EDTA, 1 ,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin egg white cystatin, and any combination thereof. The protective agent may include a phosphatase inhibitor selected from the group consisting of: calyculin A, nodularin, NIPP-1 , microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin, endothall, nodularin, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic) DMHV, sodium orthovanadate, or combinations thereof.

[0052] Optionally, the erythrocytes can be further treated by contacting with a lipoprotein during the process of preparing the blood cell analogs. The contact with lipoprotein can occur prior to fixing blood cells; and it can also occur after fixing and during storage in a suspension medium, as taught in U.S. Pat. Nos. 5,320,964, 5,512,485, 6,406,915, 6,403,377, 6,399,388, 6,221 ,668, and 6,200,500, which are herein incorporated by reference for all purposes.

[0053] One suitable example of the suspension medium includes phosphate buffered saline solution and optionally including an aqueous solution of a plasma substance. As defined herein, an aqueous solution of a plasma substance comprises an aqueous solution of a serum substance, serum substance in combination with a plasma protein, and mixtures thereof. As further defined herein, plasma protein comprises one or more of the proteins contained in plasma. Preferably, such plasma proteins include albumin, lipoproteins, globulins, fibrinogens, and mixtures thereof. Other examples of suitable medium are more fully described in U.S. Pat. Nos. 4,213,876, 4,299,726, 4,358,394, 3,873,467, 4,704,364, 5,320,964, 5,512,485, and 6,569,682, which are herein incorporated by reference for ail purposes.

[0054] In one embodiment, the linearity control system comprises a series of from about

3 to about 12 linearity control compositions described above, which are sufficient for determining linearity of measurements of one or more blood components. It is contemplated that the linearity control system can optionally include one or more single and/or multiple component control compositions prepackaged in one or more vials, and the linearity of one or more parameters corresponding to the single and/or multiple components can be determined using these compositions.

[0055] in a further aspect, the present invention provides methods of using the linearity control system of the present invention for verifying the reportable measurement range and determining linearity of measurements of one or more reportable parameters on the hematology analyzer.

[0056] While the present invention has been described in detail, these should not be construed as limitations on the scope of the present invention, but rather as an exemplification of preferred embodiments thereof. It will be apparent, however, that various modifications and changes can be made within the spirit and the scope of this invention as described in the above specification and defined in the appended claims and their legal equivalents. All patents and other publications cited herein are expressly incorporated by reference.

[0057] 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.

[0058] Unless otherwise stated, any numerical values recited herein include all values from the lower value to the upper value in increments of one unit provided that there is a separation of at least 2 units between any lower value and any higher 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.) are within the teachings of this specification. Likewise, individual intermediate values are also within the present 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 similar manner. 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.

[0059] The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes.

[0060] The term "consisting essentially of to describe a combination shall include the elements, ingredients, components or steps identified, and such other elements ingredients, components or steps that do not materially affect the basic and novel characteristics of the combination. The use of the terms "comprising" or "including" to describe combinations of elements, ingredients, components or steps herein also contemplates embodiments that consist essentially of (namely, the presence of any additional elements, ingredients, components or steps, does not materially affect the properties and/or benefits derived from the teachings; or even consist of the elements, ingredients, components or steps. [0061] 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 or metals belonging to a certain Group refer to the Periodic Table of the Elements 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 Table of the Elements using the lUPAC system for numbering groups.

[0062] It is understood that the above description is intended to be illustrative and not restrictive. Many embodiments as well as many applications besides the examples provided will be apparent to those of skill in the art upon reading the above description. 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. 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.

[0063] 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

WHAT IS CLAIMED IS:

1 . A method of making a control for resembling a predetermined concentration of an erythrocyte, reticulocyte, nucleated red blood cell, leukocyte or thrombocyte population, comprising the steps of admixing in a diluent:

at least one cellular component comprising a plurality of cells , such that upon passing through an automated hematology analyzer that analyzes the cellular component by a digital imaging technique, the cells are detected as erythrocytes, reticulocytes, nucleated red blood cells, leukocytes or thrombocytes,

and in a concentration corresponding to a known predetermined concentration.

2. The method of claim 1 , wherein the cells comprise a red blood cell analog, a reticulocyte analog, a nucleated red blood cell analog, a white blood cell analog, a platelet analog, or a combination thereof.

3. The method of claim 2, wherein the simulated red blood cell comprises a processed human erythrocyte, an animal erythrocyte, a particulated synthetic material or a combination thereof.

4. The method of any one of claims 1 -3, wherein the admixing is performed at or above room temperature .

5. The method of any one of claims 1 -4, wherein the diluent comprises water, alcohol, or a combination thereof.

6. The method of any one of the preceding claims, wherein the method includes a step of providing the plurality of cells from a first source and having a first color corresponding with a first range of grayscale values and treating the plurality of cells to alter the color to a second color corresponding to a second range of grayscale values.

7. The method of claim 6, wherein the step of treating includes subjecting the cells to a fluid that includes at least about 25% by volume carbon monoxide, alcohol, or a

combination thereof.

8. The method of claim 7, wherein the alcohol is selected from a C1 to C6 alcohol, a diol, a triol, or a combination thereof.

9. The method of any one of the preceding claims including treating the at least one cellular component with a preservative agent.

10. The method of claim 9, wherein the preservative agent includes glyceraldehyde, DL benzisothiazolinone, phthaldialdehyde, diazolidinyl urea, Imidazolidinyl urea, formaldehyde, formalin, glutaraldehyde, or a combination thereof.

1 1 . The method of any one of the preceding claims, including treating the at least one cellular component for reducing rouleaux, reducing aggregation, reducing cell stickiness, or a combination thereof.

12. The method of any one of the preceding claims, wherein the at least one cellular component is contacted with a phosphate buffered saline (PBS).

13. The method of any one of the preceding claims, including a plurality of sub- treatments separated by at least one washing step for removing a reagent from a previous step.

14. The method of any one of the preceding claims including exposing the control to illumination at a plurality of different wavelengths.

15. The method of claim 14, wherein the wavelengths of the illumination are from about 300 nm to about 900 nm.

16. The method of claim 14, wherein the wavelengths of the illumination are from about 400 nm to about 650 nm.

17. The method of any one of the preceding claims, wherein the at least one cellular component is stainable to provide for optical detection.

18. The method of any one of the preceding claims, wherein the at least one cellular component undergoes automated measurement of at least one optical density value.

19. A cellular fraction for an erythrocyte, reticulocyte, nucleated red blood cell, leukocyte, or thrombocyte linearity control, comprising:

a predetermined concentration of at least one cellular component including a plurality of cells, such that upon passing through an automated hematology analyzer that analyzes the cellular components by a digital imaging technique, the cells are detected as erythrocytes, reticulocytes, nucleated red blood cells, leukocytes, or thrombocytes, and in a concentration corresponding to a known predetermined concentration.

20. The cellular fraction of claim 19, wherein the cells comprise a red blood cell analog, a reticulocyte analog, a nucleated red blood cell analog, a white blood cell analog, a platelet analog, or a combination thereof.

21 . The cellular fraction of claim 19 or claim 20, wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 0 to about 245 for 8-bit grayscale.

22. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 50 to about 200 for 8-bit grayscale.

23. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 0 to about 100 for 8-bit grayscale.

24. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of 100 to 240 for 8-bit grayscale.

25. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of from about 20 to about 80 for 8-bit grayscale.

26. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of at least about 20 for 8-bit grayscale.

27. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of at least about 50 for 8-bit grayscale.

28. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of less than about 150 for 8-bit grayscale.

29. The cellular fraction of any one of claims 19-21 , wherein the cells when imaged using a focal plane array produces a body having a discernible boundary, a major portion of which has a grayscale reading in the range of less than about 100 for 8-bit grayscale.

30. The cellular fraction of any one of claims 19-29, wherein the visual color difference in grayscale between the cells and a non-erythrocyte is from about 0.5 to 13.5 according to test method ASTM D2616-12.

31 . The cellular fraction of any one of claims 19-30, wherein the visual color difference in grayscale between the cells and a non-erythrocyte is from about 1 to 10 according to test method ASTM D2616-12.

32. The cellular fraction of any one of claims 19-30, wherein the visual color difference in grayscale between the cells and a non-erythrocyte is from about 6 to 12 according to test method ASTM D2616-12.

33. The cellular fraction of any one of claims 19-32, wherein the cells resemble erythrocytes from a human blood sample in one or more of size and morphology.

34. The cellular fraction of any one of claims 19-33, wherein the cells have a mean corpuscular volume of from about 30 to about 150 fL, or even from about 80 fL to about 100 fL.

35. The cellular fraction of any one of claims 19-33, wherein the cells have a mean corpuscular volume of from about 20 to about 70 fL.

36. The cellular fraction of any one of claims 19-33, wherein the cells have a mean corpuscular volume of from about 1 10 to about 150 fL.

37. The cellular fraction of any one of claims 19-36, wherein the cells are preserved.

38. The cellular fraction of claim 20, wherein the red blood cell analog comprises a processed human erythrocyte, an animal erythrocyte, a particulated synthetic material or a combination thereof.

39. The cellular fraction of any one of claims 19-38, including at least one additional cellular component wherein upon passing through an automated hematology analyzer cells of the at least one second additional component are detected by the analyzer as a white blood cell.

40. The cellular fraction of any one of claims 19-39, wherein the at least one cellular component has a size prior to processing from about 80 f L to about 100 fL, or even about 150 fL to about 200 fL.

41 . The cellular fraction of any one of claims 19-40, wherein the erythrocytes, reticulocytes, nucleated red blood cells, leukocytes, or thrombocytes are fixed and/or swollen by contact with a solution containing glutaraldehyde, dimethyl sulfoxide, glyceraldehyde, DL benzisothiazolinone, phthaldialdehyde, diazolidinyl urea, Imidazolidinyl urea, formaldehyde, formalin, or any combination thereof.

42. The cellular fraction of any one of claims 19-41 , wherein the cells are alligator blood cells.

43. The cellular fraction of any one of claims 19-42, including an erythrocyte contacted by a surfactant selected from poloxamers, polyethylene glycol, octylphenol ethoxylate, Polyoxyethanyl a-tocopheryl sebacate (PTS), DL-a-tocopherol methoxypolyethylene glycol succinate, or a combination thereof.

44. The cellular fraction of any one of claims 19-43, wherein the at least one cellular component is exposed to an elevated temperature of greater than 30 ^eC, greater than 50 ^eC, or greater than 70 ^eC.

45. The cellular fraction of any one of claims 19-44, wherein the at least one cellular component includes a membrane that has been cross-linked or otherwise denatured.

46. The cellular fraction of any one of claims 19-45, wherein the at least one cellular component comprises a processed red blood cell derived from alligators, cattle, porcine, equine, newts, salamanders, turkeys, geese, chickens, sharks, cows, pigs, goats, salmon, human, or trout.

47. The cellular fraction of any one of claims 19-46, wherein the at least one cellular component includes a nucleus.

48. The cellular fraction of any one of claims 19-46, wherein the at least one cellular component is free of any nucleus.

49. A cellular fraction formed by the method of any one of claims 1 -18.

50. A linearity control made with the cellular fraction of any one of claims 19-48.

51 . The method of any one of claims 1 -18, wherein the at least one cellular component is preserved and then washed after preservation.

52. The method of any one of claims 1 -18, including a step of treating the at least one cellular component so that the cellular component appears red in color.

53. The method of any one of claims 1 -18, wherein the at least one cellular component is contacted with methylene blue.

54. The method of any one of claims 1 -18, including one or more shrinking or swelling steps.

55. A method of operating an optical imaging hematology analyzer comprising running a linearity control made using the cellular fraction of any of claims 19-48 on the analyzer.

56. The method of any one of claims 1 -18 or 51 -54, wherein the at least one cellular component is suspended in a matrix material and the matrix material has a viscosity of from about 0.01 to about 5.0 cP.

57. The method of any one of claims 1 -18 or 51 -54, including a step of diluting the control.

58. The method of any one of claims 1 -18 or 51 -54, wherein the diluent is selected from water, ethylenediaminetetraacetic acid (EDTA), magnesium gluconate, polyethylene glycol, and combinations thereof.

59. The method of any one of claims 1 -18 or 51 -54, wherein the control includes one or more of antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E-64, pepstatin, VdLPFFVdL, EDTA, 1 ,10-phenanthroline, phosphoramodon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin egg white cystatin, and any combination thereof.

60. The method of any one of claims 9-18 or 51 -54, wherein the protective agent includes a phosphatase inhibitor selected from the group consisting of: calyculin A, nodularin, NIPP-1 , microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, fostriecin, tautomycin, cantharidin, endothall, nodularin, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic) DMHV, sodium orthovanadate, and a combination thereof.