(19)
(11)EP 2 972 315 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
15.01.2020 Bulletin 2020/03

(21)Application number: 13878100.0

(22)Date of filing:  20.12.2013
(51)International Patent Classification (IPC): 
G01N 33/49(2006.01)
G01N 15/14(2006.01)
G01N 15/00(2006.01)
G01N 15/02(2006.01)
G01N 21/47(2006.01)
G01N 33/569(2006.01)
G01N 15/10(2006.01)
(86)International application number:
PCT/US2013/077197
(87)International publication number:
WO 2014/143335 (18.09.2014 Gazette  2014/38)

(54)

SYSTEM FOR OPTICAL ANALYSIS OF THE MEAN CORPUSCULAR VOLUME OF RED BLOOD CELLS

SYSTEM ZUR OPTISCHEN ANALYSE VOM MITTLEREN ZELLVOLUMEN ROTER BLUTZELLEN

SYSTÈMES POUR L'ANALYSE OPTIQUE DU VOLUME GLOBULAIRE MOYEN DES GLOBULES ROUGES


(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: 12.03.2013 US 201361778051 P

(43)Date of publication of application:
20.01.2016 Bulletin 2016/03

(60)Divisional application:
19208109.9

(73)Proprietor: ABBOTT LABORATORIES
Abbott Park, 60064 (US)

(72)Inventors:
  • WU, Jiong
    Los Gatos, California 95032 (US)
  • LIN, Ji
    San Jose, California 95129 (US)

(74)Representative: Williams Powell 
11 Staple Inn
London WC1V 7QH
London WC1V 7QH (GB)


(56)References cited: : 
WO-A1-00/58727
US-A- 5 812 419
US-A1- 2009 220 936
US-A1- 2012 282 600
WO-A2-03/021223
US-A1- 2004 018 629
US-A1- 2011 178 716
US-B2- 7 968 279
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    CROSS-REFERENCE



    [0001] This application claims the benefit of U.S. Provisional Patent Application No. 61/778,051 filed March 12, 2013.

    INTRODUCTION



    [0002] Diluent reagents have broad applications in hematology analyses, serving as, e.g., diluting reagents in the preparation of blood samples for analysis of red blood cells (RBCs), platelets (PLTs), and/or reticulocytes (RETCs). Diluent solutions also serve as, e.g., sheath solutions, rinsing solutions, maintenance solutions that prevent drying or salt precipitation on instrumentation, and the like. As such, diluent reagents for use with hematology analyzers are by far one of the largest consumable reagents in the hematology field.

    [0003] In current configurations, due to consumption rates, diluent solutions are typically stored in large containers that are heavy and difficult to manipulate. In some cases, for example, 20 liter cubitainers are used to store diluent reagents. Such containers are large and heavy, and present ergonomic issues for those who work with them. In addition, these heavy containers are expensive to ship and take up large amounts of storage space. Accordingly, there is clearly a need for more concentrated diluent reagents that would eliminate, or at least reduce the magnitude of these issues.

    [0004] The present invention addresses these and other needs

    [0005] US 2004/018629 A1 discloses a analyzer for conducting an optical analysis of erythrocytes in a sample of whole blood. A diluent reagent is added to the sample. The volumes of particles counted as erythrocytes are calculated based on forward scattered light intensity and totaled. Mean red cell volume is calculated by dividing the total value by the particle number.

    SUMMARY



    [0006] According to the present invention, there is provided a system according to claim 1 for conducting an optical mean corpuscular volume (MCV) analysis of red blood cells on a sample of whole blood. The system can be used to preserve blood cell morphology and integrity as well as provide sample integrity and optical clarity to facilitate optical analysis of blood samples. The reagents include a non-phosphate organic buffer and a sphering surfactant. The pH and osmolality of the reagents are adjusted to desired ranges. In addition, the reagents can be simply diluted with de-ionized water prior to use.

    [0007] In some embodiments, the non-phosphate organic buffer is MES, MOPS, HEPES, or imidazole. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent is at least about 0.5%. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent ranges from about 0.5% up to about 20%. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent is at least about 50 mM. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent ranges from about 50 up to about 1,500 mM. In some embodiments, the sphering surfactant is maltoside. In some embodiments, the concentration of the sphering surfactant is at least about 0.0002%. In some embodiments, the concentration of the sphering surfactant ranges from about 0.0002% up to about 2.0%. In some embodiments, the concentration of the sphering surfactant is at least about 5 mg/L. In some embodiments, the concentration of the sphering surfactant ranges from about 5 up to about 1,000 mg/L. In some embodiments, the osmolality adjustment component is sodium chloride, potassium chloride or a mixture thereof. In some embodiments, the concentration of the osmolality adjustment component is at least about 0.25%. In some embodiments, the concentration of the osmolality adjustment component ranges from about 0.25% up to about 25%. In some embodiments, the reagent further comprises an antimicrobial agent. In some embodiments, the concentration of the antimicrobial agent is at least about 0.2%. In some embodiments, the concentration of the antimicrobial agent ranges from about 0.02% up to about 0.1%. In some embodiments, the pH of the reagent ranges from about 6.0 up to about 8.0 pH units. In some embodiments, the osmolality of a IX concentration solution of the reagent ranges from about 250 up to about 350 mOsm. In some embodiments, the plurality of detectors includes one or more photomultiplier tubes and/or avalanche photo diodes (APDs). In some embodiments, the excitation source is a laser.

    [0008] In some embodiments, the subject systems further include a subsystem for diluting the reagent. In some embodiments, the subsystem is configured to mix the diluted reagent with the blood sample. In some embodiments, the subsystem is configured to incubate the blood sample with the reagent for a period of time ranging from about 1 to about 30 seconds. In some embodiments, the subsystem is configured to incubate the blood sample with the diluted reagent at a temperature ranging from about 15°C to about 50°C. In some embodiments, the subsystem is configured to incubate the blood sample with the diluted reagent at ambient temperature.

    [0009] In some embodiments, the present disclosure provides methods for performing an optical mean corpuscular volume (MCV) analysis with an automated hematology analyzer, the method including: (a) diluting a sample of whole blood with a IX concentration working solution of a hematology analysis reagent, wherein the hematology analysis reagent comprises: a non-phosphate organic buffer; a sphering surfactant; and an osmolality adjustment component, wherein the hematology reagent has sufficient optical clarity to facilitate optical analysis of the sample; (b) delivering the incubated sample from step (a) to a flow cell of the hematology analyzer; (c) exciting the incubated sample from step (b) with an excitation source as the sample traverses the flow cell; (d) collecting a plurality of light scatter signals from the excited sample; and (e) analyzing the signals collected in step (d) to determine the MCV of the sample. In some embodiments, the subject methods involve conducting a complete blood sample analysis, such as, e.g., an MCV analysis, without the use of electrical impedance measurement equipment.

    [0010] In some embodiments, the non-phosphate organic buffer is MES, MOPS, HEPES, or imidazole. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent ranges from about 0.5% up to about 20%. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent ranges from about 50 up to about 1,500 mM. In some embodiments, the sphering surfactant is maltoside. In some embodiments, the concentration of the sphering surfactant ranges from about 0.0002% up to about 2.0%. In some embodiments, the concentration of the sphering surfactant ranges from about 5 up to about 1,000 mg/L. In some embodiments, the osmolality adjustment component is sodium chloride, potassium chloride or a mixture thereof. In some embodiments, the concentration of the osmolality adjustment component ranges from about 0.25% up to about 25%. In some embodiments, the reagent further comprises an antimicrobial agent. In some embodiments, the concentration of the antimicrobial agent ranges from about 0.02% up to about 0.1%. In some embodiments, the pH of the reagent ranges from about 6.0 up to about 8.0 pH units. In some embodiments, the osmolality of a IX concentration solution of the reagent ranges from about 250 up to about 350 mOsm.

    [0011] In some embodiments, the present disclosure provides hematology reagents that include: a non-phosphate organic buffer; a sphering surfactant; and an osmolality adjustment component, wherein the hematology reagent has sufficient optical clarity to facilitate optical analysis of a blood sample.

    [0012] In some embodiments, the non-phosphate organic buffer is MES, MOPS, HEPES, or imidazole. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent is at least about 0.5%. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent ranges from about 0.5% up to about 20%. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent is at least about 50 mM. In some embodiments, the concentration of the non-phosphate organic buffer in the reagent ranges from about 50 up to about 1,500 mM. In some embodiments, the sphering surfactant is maltoside. In some embodiments, the concentration of the sphering surfactant is at leat about 0.0002%. In some embodiments, the concentration of the sphering surfactant ranges from about 0.0002% up to about 2.0%. In some embodiments, the concentration of the sphering surfactant is at least about 5 mg/L. In some embodiments, the concentration of the sphering surfactant ranges from about 5 up to about 1,000 mg/L. In some embodiments, the osmolality adjustment component is sodium chloride, potassium chloride or a mixture thereof. In some embodiments, the concentration of the osmolality adjustment component is at least about 0.25%. In some embodiments, the concentration of the osmolality adjustment component ranges from about 0.25% up to about 25%. In some embodiments, the reagent further comprises an antimicrobial agent. In some embodiments, the concentration of the antimicrobial agent is at least about 0.02%. In some embodiments, the concentration of the antimicrobial agent ranges from about 0.02% up to about 0.1%. In some embodiments, the pH of the reagent ranges from about 6.0 up to about 8.0 pH units. In some embodiments, the osmolality of a IX concentration solution of the reagent ranges from about 250 up to about 350 mOsm.

    BRIEF DESCRIPTION OF THE FIGURES



    [0013] The accompanying figures, which are incorporated herein, form part of the specification. Together with this written description, the figures further serve to explain the principles of, and to enable a person skilled in the relevant art(s), to make and use the reagents, systems and methods presented herein. In the figures, like reference numbers indicate identical or functionally similar elements.

    FIG. 1 shows a graph that correlates mean corpuscular volume (MCV) measurements taken using a IX working solution of a hematology reagent described herein to MCV measurements taken using a standard CD Sapphire reagent.

    FIG. 2 shows a graph that correlates MCV measurements taken using a IX working solution of a hematology reagent described herein to MCV measurements taken using a standard CD Ruby reagent.

    FIG. 3 is a graph that shows the impact of changes in the osmolality of a hematology reagent on optical MCV measurements.

    FIG. 4 is a graph that shows the impact of changes in the pH of a hematology reagent on optical MCV measurements.

    FIG. 5 is a graph that shows the impact of changes in the maltoside concentration of a hematology reagent on optical MCV measurements.


    DETAILED DESCRIPTION



    [0014] The present invention provides a system for conducting an optical mean corpuscular volume (MCV) analysis of red blood cells on a sample of whole blood that can be used to preserve blood cell morphology and uniformity as well as provide sample integrity and optical clarity to facilitate optical analysis of blood samples. The subject reagents include a non-phosphate organic buffer and a sphering surfactant. The pH and osmolality of the reagents are adjusted to desired ranges. In addition, the reagents can be simply reconstituted with de-ionized water prior to use.

    [0015] Red blood cells (RBCs) are the most common type of blood cell, and generally have the shape of a bi-concave disk that is flattened and compressed in the center. Quantification of RBCs in a sample of blood is typically achieved using an impedance detector that measures changes in the electrical resistance of the sample as the sample passes through a small aperture. As each individual RBC passes through the aperture, a corresponding change in impedance is detected, and the information is used to count the number of RBCs in the sample. Since impedance measurements do not involve optical data collected from the cells, the shape of the RBCs is not critical to this analysis.

    [0016] Hematological analysis systems and methods generally rely on both optical and electrical impedance measurements to analyze a blood sample and provide a differential analysis of the cells therein. For example, many hematology analyzers perform electrical impedance measurements on a sample to determine the number of RBCs and PLTs that are present in the sample, and also perform optical analyses to, e.g., quantify white blood cells (WBCs) and/or other components in the blood, and to provide additional optical analysis of RBCs and PLTs. Accordingly, most hematology analyzers must include both optical data collection components, as well as electrical impedance measurement components to provide a complete analysis of a blood sample.

    [0017] The hematology analysis reagents, systems and methods according to the present disclosure provide for optical analysis of blood samples and obviate the need for hematology analyzers to include electrical impedance measurement components. As such, the subject hematology analysis reagents, systems and methods can be used to carry out a complete analysis of a blood sample using only optical data collection components, e.g., without using electrical impedance measurements.

    HEMATOLOGY ANALYSIS REAGENTS



    [0018] The hematology analysis reagents provided herein are generally designed to facilitate complete analysis of a blood sample without the use of electrical impedance components. For example, the hematology analysis reagents provided herein provide a number of features that facilitate optical analysis of RBCs, and therefore obviate the need for electrical impedance measurements. The subject hematology reagents provide optical clarity that is necessary for optical analysis of blood samples, and also provide for sphering of RBCs so that they can be analyzed using optical techniques. The subject hematology reagents can also be highly concentrated while still maintaining their functional characteristics, which facilitates improved manufacturability, storage and handling of the reagents.

    Non-Phosphate Organic Buffer



    [0019] The subject hematology reagents generally include one or more non-phosphate organic buffers. The use of these buffers provides enhanced solubility of other components of the reagent, such as, e.g., sodium chloride as a primary osmolyte. Phosphate-based buffer components, such as phosphate buffered saline (PBS), generally cause solubility issues at high concentrations. Non-phosphate organic buffers, in contrast, are generally inert to other reagent components, such as salts, and therefore allow more concentrated reagents to be formulated while still maintaining functional properties, such as, e.g., optical clarity. Non-phosphate organic buffers in accordance with some embodiments of the invention generally have an effective buffering capacity between pH 6.0 and 8.0. Examples of suitable non-phosphate organic buffers include, but are not limited to, 2-(N-morpholine) ethane sulfonic acid (MES) buffer, 3-(N-morpholine) propane sulfonic acid (MOPS) buffer, imidazole, and N-(2-hydroxyethyl) piperazine-N'-(2-ethane sulfonic acid) (HEPES) buffer.

    [0020] In some embodiments, the concentration of the non-phosphate organic buffer in the hematology reagent ranges from about 50, up to about 75, up to about 100, up to about 125, up to about 150, up to about 175, up to about 200, up to about 225, or up to about 250, up to about 275, up to about 300, up to about 325, up to about 350, up to about 375, up to about 400, up to about 425, up to about 450, up to about 475, up to about 500, up to about 525, up to about 550, up to about 575, up to about 600, up to about 625, up to about 650, up to about 675, up to about 700, up to about 725, up to about 750, up to about 775, up to about 800, up to about 825, up to about 850, up to about 875, up to about 900, up to about 925, up to about 950, up to about 1000, up to about 1050, up to about 1100, up to about 1150, up to about 1200, up to about 1250, up to about 1300, up to about 1350, up to about 1400, up to about 1450, up to about 1500 mM or more.

    [0021] In some embodiments, the concentration of the non-phosphate organic buffer in the hematology reagent ranges from about 0.5%, up to about 1.0%, up to about 1.5%, up to about 2.0%, up to about 2.5%, up to about 3.0%, up to about 3.5%, up to about 4.0%, up to about 4.5%, up to about 5.0%, up to about 5.5%, or up to about 6.0%, up to about 6.5%, up to about 7.0%, up to about 7.5%, up to about 8.0%, up to about 8.5%, up to about 9.0%, up to about 9.5%, up to about 10.0%, up to about 10.5%, up to about 11.0%, up to about 11.5%, up to about 12.0%, up to about 12.5%, up to about 13.0%, up to about 13.5%, up to about 14.0%, up to about 14.5%, up to about 15.0%, up to about 15.5%, up to about 16.0%, up to about 16.5%, up to about 17.0%, up to about 17.5%, up to about 18.0%, up to about 18.5%, up to about 19.0%, up to about 19.5%, up to about 20.0% or more.

    Sphering Surfactant



    [0022] The subject hematology reagents generally include one or more sphering surfactants that sphere RBCs in the blood sample to facilitate their optical analysis. The use of such surfactants sustains a sphered cell morphology in diluted blood samples and facilitates optical analyses, e.g., optical mean corpuscular volume (MCV) analysis. Examples of suitable sphering surfactants include, but are not limited to, N-dodecyl-B-D-maltoside (maltoside).

    [0023] In some embodiments, the concentration of the sphering surfactant in the hematology reagent ranges from about 0.0002%, up to about 0.0005%, up to about 0.001%, up to about 0.005%, up to about 0.01%, up to about 0.05%, up to about 0.1%, up to about 0.5%, up to about 1.0%, up to about 1.25%, up to about 1.5%, up to about 1.75%, or up to about 2.0% or more. In some embodiments, the concentration of the sphering surfactant ranges from about 5, up to about 25, up to about 50, up to about 75, up to about 100, up to about 200, up to about 300, up to about 400, up to about 500, up to about 600, up to about 700, up to about 800, up to about 900, up to about 1,000 mg/L or more.

    Additional Components



    [0024] Hematology reagents in accordance with embodiments of the invention may contain additional components, such as, e.g., chelating reagents, salts or osmolytes. For example, in some embodiments, a hematology reagent may contain a chelating reagent to, e.g., prevent platelet clumping and/or aggregation. An example of a suitable chelating reagent is ethylenediaminetetraacetate (EDTA) or EDTA di-sodium salt.

    [0025] In some embodiments, a chelating reagent may be present in a hematology reagent at a concentration ranging from about 0.1%, up to about 0.2%, up to about 0.3%, up to about 0.4%, up to about 0.5% or more.

    [0026] In some embodiments, a hematology reagent may contain an osmolality adjusting component, such as, e.g., a salt. The inclusion of one or more salts in the hematology reagent serves to adjust the osmolality of the reagent for optimum performance. Suitable examples of salts include, but are not limited to, sodium chloride and potassium chloride.

    [0027] In some embodiments, an osmolality adjusting component (e.g., a salt) may be present in a hematology reagent at a concentration ranging from about 0.25%, up to about 0.5%, up to about 0.75%, up to about 1%, up to about 5%, up to about 10%, up to about 15%, up to about 20%, or up to about 25% or more.

    [0028] Hematology reagents in accordance with some embodiments of the invention may have a pH value that is adjusted for optimum performance of the reagent. For example, in some embodiments, a hematology reagent may have a pH value ranging from about 6.0, up to about 6.25, up to about 6.5, up to about 6.75, up to about 7.0, up to about 7.25, up to about 7.5, up to about 7.75, up to about 8.0 pH units. In some embodiments, the pH of a hematology reagent may be adjusted using standard pH adjustment reagents, e.g., concentrated acids or bases.

    [0029] Hematology reagents in accordance with some embodiments of the invention may have an osmolality that is adjusted for optimum performance of the reagent. Prior to its dilution to a working concentration, hematology reagents in accordance with some embodiments may have an osmolality that is hypertonic. After dilution of the hematology reagent to a IX working concentration, in some embodiments the hematology reagent may have an osmolality ranging from about 250, up to about 260, up to about 270 , up to about 280, up to about 290, up to about 300, up to about 310, up to about 320, up to about 330, up to about 340, up to about 350 mOsm or more.

    [0030] In some embodiments, a hematology reagent may include at least one preservative and/or at least one antimicrobial agent to prevent microbial growth in the reagent. Suitable examples of antimicrobial agents include, but are not limited to, Triadine™ or equivalents thereof. In some embodiments, the concentration of the antimicrobial reagent ranges from about 0.02%, up to about 0.04%, up to about 0.06%, up to about 0.08%, up to about 0.1% or more.

    [0031] In some embodiments, the concentrations of the various components in the hematology reagent are balanced and optimized to facilitate the incorporation of only minimal quantities of certain components while still maintaining the functional properties of the reagent. For example, in some embodiments, the concentration of salts and non-phosphate organic buffers included in the reagent facilitate the use of lower quantities of the sphering surfactant. This balancing of the components helps to reduce the overall cost of the reagent and improve the manufacturability of the reagent.

    [0032] In some embodiments, the concentrations of the various components in the hematology reagent are balanced to facilitate handling of the reagent. For example, in some embodiments, the concentrations of each component are carefully controlled so that the reagent maintains its functional properties and maintains solubility of the components, even when the reagent is subjected to one or more freeze/thaw cycles.

    Example Formulations:



    [0033] Various example formulations of the subject hematology reagents are provided below. The formulations provided below merely serve as examples, and are in no way limiting. Any of a variety of combinations of the components described herein can be utilized in hematology reagents in accordance with embodiments of the invention.

    Example Formulation 1:



    [0034] 
    ComponentConcentration
    EDTA di-sodium salt 0.30%
    Potassium Chloride 0.45%
    Sodium Chloride 15.70%
    Triadine 10 0.06%
    N-Dodecyl-B-D, Maltoside 0.02%
    Imidazole 0.60%
    The above formulation can be diluted by 15X to form a IX working reagent.

    Example Formulation 2:



    [0035] 
    ComponentConcentration
    EDTA di-sodium salt 0.30%
    Potassium Chloride 0.45%
    Sodium Chloride 15.70%
    Triadine 10 0.06%
    N-Dodecyl-B-D, Maltoside 0.02%
    MES 2.30%
    The above formulation can be diluted by 15X to form a IX working reagent.

    Example Formulation 3:



    [0036] 
    ComponentConcentration
    EDTA di-sodium salt 0.30%
    Potassium Chloride 0.45%
    Sodium Chloride 15.70%
    Triadine 10 0.06%
    N-Dodecyl-B-D, Maltoside 0.02%
    HEPES 3.50%
    The above formulation can be diluted by 15X to form a IX working reagent.

    Example Formulation 4:



    [0037] 
    ComponentConcentration
    EDTA di-sodium salt 0.40%
    Potassium Chloride 0.30%
    Sodium Chloride 21.24%
    Triadine 10 0.06%
    N-Dodecyl-B-D, Maltoside 0.02%
    Imidazole 0.80%
    The above formulation can be diluted by 20X to form a IX working reagent.

    Example Formulation 5 (A-I):



    [0038] A series of reagent formulations (A-I) are provided below, wherein the concentration of sodium chloride was varied as indicated:
    ComponentABCDEFGHI
    EDTA di-sodium salt 0.40% 0.40% 0.40% 0.40% 0.40% 0.40% 0.40% 0.40% 0.40%
    Potassium Chloride 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30%
    Sodium Chloride 20.39% 20.60% 20.82% 21.03% 21.24% 21.45% 21.66% 21.88% 22.09%
    Triadine 10 0.06% 0.06% 0.06% 0.06% 0.06% 0.06% 0.06% 0.06% 0.06%
    N-Dodecyl-B-D, Maltoside 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02%
    Imidazole 0.80% 0.80% 0.80% 0.80% 0.80% 0.80% 0.80% 0.80% 0.80%
    Osmolality (mOsmo/kg) of 1X working reagent 313 317 319 322 322 325 329 331 335

    Example Formulation 6 (A-G):



    [0039] A series of reagent formulations (A-G) are provided below, wherein the pH value of the reagent varied as indicated. Concentrated HCl was added to each formulation in a sufficient quantity to adjust the pH to the values indicated below.
    ComponentABCDEFG
    EDTA di-sodium salt 0.40% 0.40% 0.40% 0.40% 0.40% 0.40% 0.40%
    Potassium Chloride 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30%
    Sodium Chloride 21.24% 21.24% 21.24% 21.24% 21.24% 21.24% 21.24%
    Triadine 10 0.06% 0.06% 0.06% 0.06% 0.06% 0.06% 0.06%
    N-Dodecyl-B-D, Maltoside 0.02% 0.02% 0.02% 0.02% 0.02% 0.02% 0.02%
    Imidazole 0.80% 0.80% 0.80% 0.80% 0.80% 0.80% 0.80%
    pH 6.95 7.20 7.35 7.45 7.55 7.70 7.95

    Example Formulation 7 (A-G):



    [0040] A series of reagent formulations (A-G) are provided below, wherein the concentration of maltoside in the reagent varied as indicated:
    ComponentABCDEFG
    EDTA di-sodium salt 0.40% 0.40% 0.40% 0.40% 0.40% 0.40% 0.40%
    Potassium Chloride 0.30% 0.30% 0.30% 0.30% 0.30% 0.30% 0.30%
    Sodium Chloride 21.24% 21.24% 21.24% 21.24% 21.24% 21.24% 21.24%
    Triadine 10 0.06% 0.06% 0.06% 0.06% 0.06% 0.06% 0.06%
    N-Dodecyl-B-D, Maltoside -5% -2.5% -1% 0.0% +1% +2.5% +5%
    Imidazole 0.80% 0.80% 0.80% 0.80% 0.80% 0.80% 0.80%

    SYSTEMS AND METHODS



    [0041] The hematology reagents disclosed herein generally find use in the optical analysis of blood samples using hematology analysis systems and related methods. In certain embodiments, the subject hematology reagents find use in hematology analysis systems and methods that are designed to optically measure RBCs in a sample of blood. For example, in some embodiments the subject systems may be used to perform a complete blood sample analysis without the use of electrical impedance measurements, e.g., using only optical measurements.

    [0042] In some embodiments, the subject hematology reagents are diluted with a suitable reagent, e.g., de-ionized water, prior to their use in analyzing a blood sample. For example, in some embodiments, a subject hematology reagent may be diluted by a factor of about 2X, up to about 5X, up to about 10X, up to about 15X, up to about 20X, or up to about 25X to form a working solution of the reagent that has a concentration of IX.

    [0043] In some embodiments, a subject hematology reagent may be diluted to an appropriate concentration using simple mixing techniques, for example, wherein the reagent is mixed with de-ionized water in a container of a suitable size and mechanically mixed, stirred, etc. In some embodiments, mixing of the hematology reagent with de-ionized water can be accomplished using, e.g., an electronic mixing device.

    [0044] In some embodiments, a working solution of the reagent may be created prior to its use in a hematology analyzer. For example, in some embodiments a working solution having a concentration of IX of the hematology reagent is formed as described above, and the IX working solution is then introduced into or fluidly coupled to a hematology analyzer for use in analyzing a blood sample. According to the claimed invention, a concentrated form of the subject hematology reagents is fluidly coupled to the hematology analyzer, and the analyzer may perform a dilution of the hematology reagent prior to or concurrent with the analysis of a blood sample.

    [0045] In certain embodiments, a IX concentration working solution of a subject hematology reagent is mixed with a blood sample, and the blood sample is analyzed on an automated hematology analyzer that generates a plurality of optical data from the sample be directing a light source towards the sample as it is passed through a flow cell. In some embodiments, the hematology analyzer may include a processor containing instructions that, when executed by the processor, cause the hematology analyzer to carry out a series of steps that involve moving a sample through the flow cell of the analyzer, directing light towards the flow cell, gathering a plurality of optical data from the sample, and analyzing the optical data to determine, e.g., an MCV measurement from the sample based on the optical data.

    [0046] Examples, not being part of the claimed invention, of methods of analyzing a blood sample to determine the MCV of the sample using optical techniques involve: (a) contacting a blood sample with a IX working solution of a reagent that comprises at least one non-phosphate organic buffer, at least one sphering surfactant, and one or more osmolality adjustment components and incubating the blood sample with the reagent for a period of time ranging from about 1 to about 30 seconds at a temperature ranging from about 15°C up to about 50 °C, such as, e.g., ambient temperature; (b) delivering the sample from step (a) to a flow cell of a hematology analyzer; (c) exciting the sample from step (b) with an excitation source as the sample traverses the flow cell; (d) collecting a plurality of light scatter signals from the excited sample; and (e) analyzing the signals collected in step (d) to the determine the MCV of the sample.

    [0047] Examples not being part of the claimed invention:

    Example 1: MCV measurement comparison using different reagents



    [0048] MCV measurements were taken on a normal blood sample using a subject hematology reagent that was diluted from 15X or 20X to form a solution of IX concentration. The MCV measurements were compared with MCV measurements that were taken on the same normal blood sample using a CD Sapphire or CD Ruby reagent. The results are shown in FIG. 1 and FIG. 2. The results show that the subject hematology reagents perform equally well using either impedance or optical detection systems.

    Example 2: Impact of Osmolality Variation on Optical MCV Measurements



    [0049] A series of reagents was formulated having different sodium chloride concentrations to adjust the final osmolality of the working reagent when used in a hematology analyzer to measure MCV. The concentrations of the components in the reagents are shown in example formulations 5A-I. The reagents were then used to measure the MCV of a normal blood sample on a hematology analyzer. The results are shown in FIG. 3, and indicate that the formulations function as expected over the tested osmolality range.

    Example 3: Impact of pH Variation on Optical MCV Measurements



    [0050] A series of reagents was formulated having different pH values. The concentrations of the components in the reagents are shown in example formulations 6A-G. The reagents were then used to measure the MCV of a normal blood sample on a hematology analyzer. The results are shown in FIG. 4, and indicate that the formulations function as expected over the tested pH range.

    Example 4: Impact of Maltoside Variation on Optical MCV Measurements



    [0051] A series of reagents was formulated having different maltoside concentrations. The concentrations of the components in the reagents are shown in example formulations 7A-G. The reagents were then used to measure the MCV of a normal blood sample on a hematology analyzer. The results are shown in FIG. 5, and indicate that the formulations function as expected over the tested maltoside concentration range.

    [0052] The foregoing description of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Other modifications and variations within the scope of the appended claims may be possible in light of the above teachings. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, and to thereby enable others skilled in the art to best utilize the invention in various embodiments and various modifications as are suited to the particular use contemplated. It is intended that the appended claims be construed to include other alternative embodiments of the invention.

    [0053] The above Detailed Description refers to the accompanying drawings that illustrate one or more exemplary embodiments. Other embodiments are possible. Modifications may be made to the embodiment described without departing from the scope of the present invention. Therefore, the Detailed Description is not meant to be limiting. Further, the Summary and Abstract sections may set forth one or more, but not all exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.


    Claims

    1. A system for conducting an optical mean corpuscular volume (MCV) analysis of red blood cells on a sample of whole blood, the system comprising:

    (a) a hematology analyzer, the hematology analyzer comprising:

    a flow cell;
    an excitation source positioned to excite particles within the blood sample as it is passed through the flow cell;
    a plurality of detectors including (1) an axial light loss detector positioned to measure axial light loss from the excited blood sample, (2) an intermediate angle scatter detector positioned to measure intermediate angle scatters from the excited blood sample, (3) a polarized side scatter detector positioned to measure large angle polarized side scatters from the excited blood sample, (4) a depolarized side scatter detector positioned to measure large angle depolarized side scatters from the excited blood sample; and

    a processor configured to:

    (I) receive the measurements of (1) axial light loss, (2) intermediate angle scatters, (3) large angle polarized side scatters, (4) large angle depolarized side scatters, and

    (II) perform an MCV analysis of red blood cells of the blood sample, based on a plurality of optical data received from the detectors and without the use of electrical impedance components; and

    (b) a hematology reagent fluidly coupled to the hematology analyzer for optical MCV analysis of red blood cells, the hematology reagent comprising:

    a non-phosphate organic buffer;

    a sphering surfactant; and

    an osmolality adjustment component,

    wherein the hematology reagent has sufficient optical clarity to facilitate optical MCV analysis of the sample.


     
    2. The system according to Claim 1, wherein the non-phosphate organic buffer is 2-(N-morpholine) ethane sulfonic acid (MES), 3-(N-morpholine) propane sulfonic acid (MOPS), N-(2-hydroxyethyl) piperazine-N'-(2-ethane sulfonic acid) (HEPES), or imidazole.
     
    3. The system according to any of the preceding claims, wherein the concentration of the non-phosphate organic buffer in the reagent is at least 0.5%.
     
    4. The system according to any of the preceding claims, wherein the concentration of the non-phosphate organic buffer in the reagent is at least 50 mM.
     
    5. The system according to any of the preceding claims, wherein the sphering surfactant is maltoside.
     
    6. The system according to any of the preceding claims, wherein the concentration of the sphering surfactant is at least 0.0002%.
     
    7. The system according to any of the preceding claims, wherein the concentration of the sphering surfactant ranges from 0.0002% to 2.0%.
     
    8. The system according to any of the preceding claims, wherein the concentration of the sphering surfactant is at least 5 mg/L.
     
    9. The system according to any of the preceding claims, wherein the concentration of the sphering surfactant ranges from 5 to 1,000 mg/L.
     
    10. The system according to any of the preceding claims, wherein the osmolality adjustment component is sodium chloride, potassium chloride or a mixture thereof.
     
    11. The system according to any of the preceding claims, wherein the concentration of the osmolality adjustment component is at least 0.25%.
     
    12. The system according to any of the preceding claims, wherein the concentration of the osmolality adjustment component ranges from 0.25% to 25%.
     
    13. The system according to any of the preceding claims, wherein the pH of the reagent ranges from 6.0 to 8.0 pH units.
     
    14. The system according to any of the preceding claims, wherein the osmolality of a IX concentration solution of the reagent ranges from 250 to 350 mOsm.
     
    15. The system according to any of the preceding claims, wherein the plurality of detectors include one or more photomultiplier tubes and/or avalanche photo diodes (APDs).
     
    16. The system according to any of the preceding claims, wherein the excitation source is a laser.
     
    17. The system according to any of the preceding claims, further comprising a subsystem for diluting the reagent.
     
    18. The system according to Claim 17, wherein the subsystem is configured to mix the diluted reagent with the blood sample.
     
    19. The system according to Claim 18, wherein the subsystem is configured to incubate the blood sample with the reagent for a period of time ranging from 1 to 30 seconds.
     
    20. The system according to Claim 18 or 19, wherein the subsystem is configured to incubate the blood sample with the diluted reagent at a temperature ranging from 15°C to 50°C.
     
    21. The system according to Claim 18, 19, or 20, wherein the subsystem is configured to incubate the blood sample with the diluted reagent at ambient temperature.
     


    Ansprüche

    1. System zum Durchführen einer optischen Analyse des mittleren korpuskulären Volumens (MCV) roter Blutkörperchen an einer Probe von Vollblut, wobei das System aufweist:

    a) einen Hämatologie-Analysator, wobei der Hämatologie-Analysator aufweist:

    eine Durchflusszelle;

    eine Anregungsquelle, die dazu angeordnet ist, Teilchen innerhalb der Blutprobe anzuregen, während diese die Durchflusszelle passiert;

    mehrere Detektoren, die aufweisen: 1) einen Axiallichtverlustdetektor, der dazu angeordnet ist, einen axialen Lichtverlust an der angeregten Blutprobe zu messen, 2) einen Zwischenwinkelstreuungsdetektor, der dazu angeordnet ist, Zwischenwinkelstreuungen aus der angeregten Blutprobe zu messen, 3) einen Detektor für polarisierte Seitwärtsstreuungen, der dazu angeordnet ist, polarisierte Seitwärtsstreuungen mit großem Winkel aus der angeregten Blutprobe zu messen, 4) einen Detektor für depolarisierte Seitwärtsstreuungen, der dazu angeordnet ist, depolarisierte Seitwärtsstreuungen mit großem Winkel aus der angeregten Blutprobe zu messen; und

    einen Prozessor, der dazu konfiguriert ist:

    I) die Messungen zu empfangen von 1) axialem Lichtverlust, 2) Zwischenwinkelstreuungen, 3) polarisierten Seitwärtsstreuungen mit großem Winkel, 4) depolarisierten Seitwärtsstreuungen mit großem Winkel, und

    II) eine MCV-Analyse an roten Blutkörperchen der Blutprobe auf Basis mehrerer von den Detektoren empfangener optischer Daten und ohne die Verwendung von elektrischer Impedanz-Komponenten durchzuführen; und

    b) ein Hämatologiereagenz, das fluidisch mit dem Hämatologie-Analysator gekoppelt ist, für eine optische MCV-Analyse roter Blutkörperchen, wobei das Hämatologiereagenz aufweist:

    einen organischen Puffer, der kein Phosphat ist;

    eine aufkugelnde oberflächenaktive Substanz; und

    eine Komponente zur Einstellung der Osmolalität,
    wobei das Hämatologiereagenz genügend optische Klarheit aufweist, um eine optische MCV-Analyse der Probe zu ermöglichen.


     
    2. System gemäß Anspruch 1, wobei der organische Puffer, der kein Phosphat ist, 2-(N-Morpholino)ethansulfonsäure (MES), 3-(N-Morpholino)propansulfonsäure (MOPS), N-(2-Hydroxyethyl)piperazin-N'-(2-ethansulfonsäure) (HEPES), oder Imidazol ist.
     
    3. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration des organischen Puffers, der kein Phosphat ist, in dem Reagenz mindestens 0,5% beträgt.
     
    4. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration des organischen Puffers, der kein Phosphat ist, in dem Reagenz mindestens 50 mM beträgt.
     
    5. System gemäß einem der voranghenden Ansprüche, wobei die aufkugelnde oberflächenaktive Substanz ein Maltosid ist.
     
    6. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration der aufkugelnden oberflächenaktiven Substanz mindestens 0,0002% beträgt.
     
    7. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration der aufkugelnden oberflächenaktiven Substanz in einem Bereich von 0,0002% bis 2,0% liegt.
     
    8. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration der aufkugelnden oberflächenaktiven Substanz mindestens 5 mg/l beträgt.
     
    9. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration der aufkugelnden oberflächenaktiven Substanz in einem Bereich von 5 bis 1.000 mg/l liegt.
     
    10. System gemäß einem der voranghenden Ansprüche, wobei die Komponente zur Einstellung der Osmolalität Natriumchlorid, Kaliumchlorid oder eine Mischung daraus ist.
     
    11. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration der Komponente zur Einstellung der Osmolalität weniger als 0,25% beträgt.
     
    12. System gemäß einem der voranghenden Ansprüche, wobei die Konzentration der Komponente zur Einstellung der Osmolalität in einem Bereich von 0,25% bis 25% liegt.
     
    13. System gemäß einem der voranghenden Ansprüche, wobei der pH-Wert des Reagenzes in einem Bereich von 6,0 bis 8,0 pH-Einheiten liegt.
     
    14. System gemäß einem der voranghenden Ansprüche, wobei die Osmolalität einer Lösung des Reagenzes einer 1X Konzentration in einem Bereich von 250 bis 350 mosm liegt.
     
    15. System gemäß einem der voranghenden Ansprüche, wobei die mehreren Detektoren einen oder mehrere Photomultiplierröhren und/oder Avalanche-Photodioden (APD) aufweisen.
     
    16. System gemäß einem der voranghenden Ansprüche, wobei die Anregungsquelle ein Laser ist.
     
    17. System gemäß einem der voranghenden Ansprüche, das ferner ein Untersystem zum Verdünnen des Reagenzes aufweist.
     
    18. System gemäß Anspruch 17, wobei das Untersystem dazu konfiguriert ist, das verdünnte Reagenz mit der Blutprobe zu mischen.
     
    19. System gemäß Anspruch 18, wobei das Untersystem dazu konfiguriert ist, die Blutprobe mit dem Reagenz über einen Zeitraum zu inkubieren, der von 1 bis 30 Sekunden beträgt.
     
    20. System gemäß Anspruch 18 oder 19, wobei das Untersystem dazu konfiguriert ist, die Blutprobe mit dem verdünnten Reagenz bei einer Temperatur von 15°C bis 50°C zu inkubieren.
     
    21. System gemäß Anspruch 18, 19 oder 20, wobei das Untersystem dazu konfiguriert ist, die Blutprobe mit dem verdünnten Reagenz bei Umgebungstemperatur zu inkubieren.
     


    Revendications

    1. Système pour conduire une analyse optique de volume globulaire moyen (VGM) de globules rouges sur un échantillon de sang total, le système comprenant :

    (a) un analyseur d'hématologie, l'analyseur d'hématologie comprenant :

    une cuve à circulation ;

    une source d'excitation positionnée pour exciter des particules à l'intérieur de l'échantillon de sang alors qu'il est amené à passer à travers la cuve à circulation ;

    une pluralité de détecteurs comprenant (1) un détecteur de perte de lumière axiale positionné pour mesurer une perte de lumière axiale à partir de l'échantillon de sang excité, (2) un détecteur de diffusion en angle intermédiaire positionné pour mesurer des diffusions en angle intermédiaire à partir de l'échantillon de sang excité, (3) un détecteur de diffusion latérale polarisée positionné pour mesurer des diffusions latérales polarisées à grand angle à partir de l'échantillon de sang excité, (4) un détecteur de diffusion latérale dépolarisée positionné pour mesurer des diffusions latérales dépolarisées à grand angle à partir de l'échantillon de sang excité ; et

    un processeur configuré pour :

    (I) recevoir les mesures de (1) perte de lumière axiale, (2) diffusions en angle intermédiaire, (3) diffusions latérales polarisées à grand angle, (4) diffusions latérales dépolarisées à grand angle, et

    (II) effectuer une analyse de VGM de globules rouges de l'échantillon de sang, sur la base d'une pluralité de données optiques reçues à partir des détecteurs et sans l'utilisation de composants d'impédance électrique ; et

    (b) un réactif d'hématologie couplé fluidiquement à l'analyseur d'hématologie pour l'analyse optique de VGM de globules rouges, le réactif d'hématologie comprenant :

    un tampon organique non phosphate ;

    un tensio-actif de sphéricité ; et

    un composant d'ajustement d'osmolalité,
    le réactif d'hématologie ayant une clarté optique suffisante pour faciliter une analyse optique de VGM de l'échantillon.


     
    2. Système selon la revendication 1, dans lequel le tampon organique non phosphate est l'acide 2-(N-morpholine)éthane sulfonique (MES), l'acide 3-(N-morpholine)-propane sulfonique (MOPS), le N-(2-hydroxyéthyl)pipérazine-N'-(2 éthane sulfonique acide) (HEPES) ou l'imidazole.
     
    3. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du tampon organique non phosphate dans le réactif est d'au moins 0,5 %.
     
    4. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du tampon organique non phosphate dans le réactif est d'au moins 50 mM.
     
    5. Système selon l'une quelconque des revendications précédentes, dans lequel le tensio-actif de sphéricité est le maltoside.
     
    6. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du tensio-actif de sphéricité est d'au moins 0,0002 %.
     
    7. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du tensio-actif de sphéricité se situe dans la plage de 0,0002 % à 2,0 %.
     
    8. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du tensio-actif de sphéricité est d'au moins 5 mg/L.
     
    9. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du tensio-actif de sphéricité se situe dans la plage de 5 à 1000 mg/L.
     
    10. Système selon l'une quelconque des revendications précédentes, dans lequel le composant d'ajustement d'osmolalité est le chlorure de sodium, le chlorure de potassium ou un mélange de ceux-ci.
     
    11. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du composant d'ajustement d'osmolalité est d'au moins 0,25 %.
     
    12. Système selon l'une quelconque des revendications précédentes, dans lequel la concentration du composant d'ajustement d'osmolalité se situe dans la plage de 0,25 % à 25 %.
     
    13. Système selon l'une quelconque des revendications précédentes, dans lequel le pH du réactif se situe dans la plage de 6,0 à 8,0 unités de pH.
     
    14. Système selon l'une quelconque des revendications précédentes, dans lequel l'osmolalité d'une solution de concentration 1 X du réactif se situe dans la plage de 250 à 350 mOsm.
     
    15. Système selon l'une quelconque des revendications précédentes, dans lequel les différents détecteurs comprennent un ou plusieurs tubes photomultiplicateurs et/ou des photodiodes à avalanche (APD).
     
    16. Système selon l'une quelconque des revendications précédentes, dans lequel la source d'excitation est un laser.
     
    17. Système selon l'une quelconque des revendications précédentes, comprenant en outre un sous-système pour diluer le réactif.
     
    18. Système selon la revendication 17, dans lequel le sous-système est configuré pour mélanger le réactif dilué avec l'échantillon de sang.
     
    19. Système selon la revendication 18, dans lequel le sous-système est configuré pour faire incuber l'échantillon de sang avec le réactif pendant une période de temps se situant dans la plage de 1 à 30 secondes.
     
    20. Système selon l'une des revendications 18 ou 19, dans lequel le sous-système est configuré pour faire incuber l'échantillon de sang avec le réactif dilué à une température se situant dans la plage de 15°C à 50°C.
     
    21. Système selon l'une des revendications 18, 19 ou 20, dans lequel le sous-système est configuré pour faire incuber l'échantillon de sang avec le réactif dilué à la température ambiante.
     




    Drawing

















    Cited references

    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