USH602H - Whole blood diluting solution - Google Patents

Whole blood diluting solution Download PDF

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Publication number
USH602H
USH602H US07/097,624 US9762487A USH602H US H602 H USH602 H US H602H US 9762487 A US9762487 A US 9762487A US H602 H USH602 H US H602H
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United States
Prior art keywords
whole blood
diluting solution
water
dispersed phase
blood sample
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Abandoned
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US07/097,624
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English (en)
Inventor
Masaaki Terashima
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Fujifilm Holdings Corp
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Fuji Photo Film Co Ltd
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Assigned to FUJI PHOTO FILM CO., LTD., NO. 210, NAKANUMA, MINAMIASHIGARA-SHI, KANAGAWA, JAPAN reassignment FUJI PHOTO FILM CO., LTD., NO. 210, NAKANUMA, MINAMIASHIGARA-SHI, KANAGAWA, JAPAN ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: TERASHIMA, MASAAKI
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a diluting solution for diluting a whole blood sample, if needed, in analyzing a target component or analyte in the quantitative analysis contained in the whole blood sample, using an element for dry analysis. More particularly, it is concerned with a diluting solution and an analytical process using said diluting solution for the quantitative analysis of diluted whole blood samples using, for example, analysis element comprising paper impregnated with a color-producing reagent, or a multilayer analysis element comprising a light-permeable, water-impermeable support having at least one reagent layer and an outermost porous layer.
  • Multilayer dry analysis elements comprising a transparent support having thereon at least one reagent layer and a porous layer, in this order, and quantitative analysis methods of aqueous liquid samples using those materials are described, e.g., in, Japanese Patent Application (OPI) Nos. 53888/74 137192/75, 140191/76, 3488/77, 131089/78, 101398/79, 90859/80, 164356/80, 24576/81, etc. (as used herein "OPI” means an "unexamined published application”), H. G. Curme et al and R. W. Spayd et al., Clinical Chemistry, vol. 24, pp. 1335-1350 (1978), Bert Walter, Anal. Chem., vol. 55, No. 4, pp. 498-514, and so on. These references describe the possibility of using non-diluted serum, blood plasma and diluted whole blood as a sample.
  • the diluting solution should be a solution having, substantially no hemolytic function, and further, does not cause agglutination of red corpuscles by dilution so that no change in fluidity of the whole blood sample occurs.
  • aqueous diluting solutions having the above characteristics
  • isotonic solutions containing inorganic salts such as, physiological saline (including physiological salt solution, Ringer's solution, etc.)
  • isotonic solutions having a viscosity adjusted properly by addition of a hydrophilic or water-soluble organic substance, such as dextran, polyvinyl pyrrolidone, albumin, etc. have generally been employed.
  • whole blood samples diluted with these aqueous diluting solutions exhibit responses different from their neat whole blood samples with respect to, e.g., color development of an analyte applied to a dry analysis element.
  • the analyte concentration calculated by multiplying the concentration derived from the calibration curve determined using neat whole blood samples, by the dilution factor is not exact.
  • the concentration-dilution factor relationship to correct the above-described error is so complicated that the dilution with the isotonic solutions described above is impractical.
  • An object of the present invention is to provide a diluting solution which can produce satisfactory results in quantitative analyses using a dry analysis element of not only normal whole blood but also abnormal blood, wherein the blood sample is diluted to a definite volume with said diluting solution. It is a further object of the present invention is to extend an applicable scope of dry analysis elements to whole blood samples, particularly, to abnormal whole blood samples.
  • the object of the present invention is to achieve analytical precision and accuracy equivalent to those attainable for normal whole blood samples, by dilution with a diluting solution of a gathered sample, in the case where the sample to be analyzed is
  • the above-described objects are attained by using as the whole blood diluting solution, a diluting solution containing a water-insoluble dispersed phase.
  • FIG. 1 and FIG. 2 are schematic diagrams for illustrating a method of analyzing a whole blood sample using a multilayer film for dry analysis.
  • FIG. 3 is a correlation graph obtained by plotting the dGlucoroder values against the (A ⁇ 4) values of Table 1.
  • triangular marks ( ⁇ ) designates data of whole blood samples having a hematocrit value of 20%
  • square marks ( ⁇ ) designates those having a hematocrit value of 40%
  • round marks (O) those having a hematocrit value of 60%.)
  • FIG. 4 is a correlation graph obtained by plotting the Glucoroder value against the whole blood (undiluted) value of Table 1.
  • the exemplified multilayer analysis element is an analysis film having a multilayer structure provided with a reagent layer 2, light-reflecting layer 3 and a porous spreading layer 4 on one side of a transparent support 1 (wherein, of course, plural reagent layers, a barrier layer, a scavenger layer, a buffer layer, a detector and so on may be interposed between the support and the spreading layer, if needed).
  • the porous spreading layer 4 is made up of a material selected from among membrane filter-form nonfibrous isotropic porous materials, porous materials made of powdery granules, textiles, paper, and so on.
  • the spreading layer is capable of a metering or spreading function such that when a drop of aqueous liquid sample is placed thereon, the aqueous liquid spreads rapidly in a circle in the horizontal direction and then penetrates in vertical direction, and the aqueous liquid is supplied to the reagent layer located thereunder in an approximately constant volume per unit area.
  • using a textile having a construction made up of fine granules and containing continuous vacant spaces therein as described in Japanese Patent Application (OPI) No. 90859/80, and the like, enables the quantitive analysis of whole blood, because those materials possess a spreading function with a respect to not only blood plasma and serum, but also, whole blood containing a concrete component.
  • a whole blood diluting solution to be used in the present invention which contains a water-insoluble dispersed phase, is described in detail below.
  • FIG. 2 illustrates schematically the phenomenon wherein a concrete component in the whole blood sample (e.g., red blood corpuscles) is filtered out by the porous spreading layer to remain on the surface and the inner part near the surface of the spreading layer, while a liquid component is spreaded by passing through the spreading layer, and further passes through the light-reflecting layer, and reaches the reagent layer.
  • a concrete component in the whole blood sample e.g., red blood corpuscles
  • the water-insoluble dispersed phase is a solid phase, it can be made up of a substance selected from a group consisting of styrene homopolymer, copolymers prepared from styrene and monomers copolymerizable with styrene, acrylate homopolymers, copolymers prepared from acrylates and monomers copolymerizable with acrylates, vinyl acetate homopolymer, copolymers prepared from vinyl acetate and monomers copolymerizable with vinyl acetate, vinyl chloride homopolymer, copolymers prepared from vinyl chloride and monomers copolymerizable with vinyl chloride, red blood corpuscles, and ghosts of red blood corpuscles.
  • the molecular weight thereof ranges preferably from 1 ⁇ 10 4 to 1 ⁇ 10 6 .
  • Preferable monomer copolymerizable with styrene, acrylate, vinyl acetate, or vinyl chloride described above is at least one monomer selected from the group consisting of acrylic acid, acrylonitrile, acrylamide, ethylene and maleic acid.
  • the water-insoluble dispersed phase is a liquid phase, it can be made up of a substance selected from a group consisting of adipates, sebacates, trimellitates and phosphates. Details of these substances are disclosed in Japanese Patent Application (OPI) No. 122956/81.
  • the alcohol moiety composing the above esters preferably includes a straight or branched alcohol having from 4 to 10 carbon atoms.
  • the diluting solution contains preferably from 10 to 50 wt %, more preferably from 15 to 30 wt % of the water-insoluble dispersed phase.
  • the undiluted whole blood sample may be diluted to preferably from 2 to 10 times, more preferably from 3 to 5 times by volume, using the diluting solution.
  • the above-described water-insoluble dispersed phase is an emulsion or a suspension having a particle size which ranges from 0.01 micron to 10 microns. So long as the phase to be dispersed can be mixed homogeneously with a water phase by a simple stirring operation, the phase may be employed in the present invention, Thus, the dispersed phase is not always required to be a stable dispersion. However, it is more desirable that the dispersed phase assumes the form of stable suspension or emulsion.
  • known additive such as, surface active substances, defoaming agents, antiseptics, etc. can be added to the aqueous diluting solution so long as the addition does not cause interference with the intended analyses.
  • Organic solvents which can be used in the invention include alcohols, such as, methanol, ethanol, benzyl alcohol, etc. Other organic liquid substances can be also added to the aqueous diluting solution.
  • antiseptics As suitable examples of antiseptics, mention may be made of parachlorophenol derivatives and benzothiazole derivatives as described in Japanese Patent Application Nos. 58765/86 and 89348/86.
  • a glycolysis inhibitor, agglutination inhibitor and like additives are generally added to whole blood taken from a human or animal body immediately after blood sampling, except when the sampled whole blood is immediately subjected to analysis.
  • Dilution can be carried out by (1) adding an aqueous diluting solution to whole blood, (2) adding whole blood to an aqueous diluting solution, or (3) pouring both whole blood and an aqueous diluting solution almost simultaneously into a third vessel.
  • the resulting mixture is preferably submitted to gentle stirring or shaking in order to form a homogeneous mixture of the aqueous diluting solution with the plasma component in the whole blood, and particularly, the concrete component in the whole blood.
  • a droplet forming instrument such as, a micropipette
  • a definite volume of whole blood with the same micropipette to produce a mixture of the components.
  • a drop of diluted whole blood sample is then placed on the porous spreading layer of a dry analysis material and incubated, if necessary.
  • the optical density of the color developed area is measured by a reflex photometry, and a content of the analyte in the whole blood sample is determined according to the principle of the colorimetric method.
  • the analyte content in the diluted whole blood sample is determined first.
  • analyte content in the undiluted whole blood sample is determined at once by measuring the optical density of the color-developed region and using the same calibration curve obtained from the undiluted whole blood sample, analyte content in the undiluted whole blood sample can be determined only by multiplication of the content in the diluted sample by the dilution factor.
  • the dilution factor is set for 2, 3, 4 or 5, the multiplication becomes very easy.
  • the hematocrit value of whole blood varies widely with the individual and therefore, a proportion of the volume occupied by blood plasma in whole blood fluctuates widely depending on the hematocrit value, too. Nevertheless, an analyte content in the undiluted whole blood sample can be derived from the analyte content in the diluted whole blood sample by using only the entire volume of the whole blood sample and the dilution factor which is a remarkable characteristic of the method of the present invention.
  • application of the whole blood diluting solution of the present invention is not confined to materials for dry colorimetric analysis. It is also possible to apply it to analysis methods which treat whole blood as a sample and use a chemical sensor utilizing an oxygen electrode, a carbon dioxide electrode, a pH electrode, an enzyme electrode, a filed effect transistor (FET) or so on.
  • the glucose concentration in whole blood was determined in the following manner, in which a diluting solution for diluting whole blood and a quantitative multilayer analysis film for glucose were employed.
  • Fresh human blood to which heparin was added immediately after blood-gathering was centrifuged to separate it into a plasma component and a corpuscle component. These two components were taken out of the apparatus separately, and then mixed in various ratios to reprepare whole blood samples having hematocrit values within the range of 20% to 60%. Further, glucose was added and dissolved into each whole blood sample in an amount necessary to adjust the glucose concentration to about 100 to 1600 mg/dl. Thus, 30 kinds of whole blood samples as shown in Table 1, which differed in hematocrit value and glucose concentration, were prepared.
  • a diluted whole blood sample was prepared by adding 300 microliter of the aqueous diluting solution to 100 microliter of the above-described whole blood sample to dilute the whole blood sample exactly 4 times by volume ratio.
  • a coating solution for forming a reagent layer which had the following composition, was coated so as to have a dry thickness of 15 microns, and dried.
  • a coating solution for forming a light shielding layer On the reagent layer, was coated a coating solution for forming a light shielding layer, which had the following position, so as to have a dry thickness of 7 microns, and dried.
  • an adhesive layer having the following composition was coated so as to have a dry thickness of 2 ⁇ m, and dried.
  • the adhesive layer was moistened with water in a quantity of 30 g/m 2 , and cotton broadcloth was adhered to it by applying light pressure thereon, followed by drying.
  • the glucose analyzing film made in the above-described manner was cut into pieces measuring 15 mm ⁇ 15 mm in size, and each piece was put on a thermoplastic resin flame measuring 24 mm by 28 mm in size.
  • each whole blood sample was centrifuged, and the resulting blood plasma was examined for glucose concentration using a Glucoroder (made by Shino Test Co., Ltd.), or a glucose analyzer based on an enzyme electrode process.
  • the results obtained are set forth at the second column from the left.
  • Hct in the table refers to a hematocrit value.
  • the conventional method in which glucose concentration is determined through putting a drop of undiluted whole blood sample on a multilayer material for dry analysis has a tendency to produce glucose concentration values lower than those obtained by a Glucoroder based on an enzyme electrode process.
  • This latter method has been prevailingly used for measurement of glucose concentration in blood when whole blood samples having high glucose concentrations are analyzed. This tendency becomes more pronounced as the hematocrit value becomes higher.
  • the glucose concentration value determined with the method of the present invention in which a whole blood sample, diluted 4 times with the whole blood diluting solution of the present invention, is put in droplet form on a multilayer material for dry analysis provided data, agree well with those obtained by the enzyme electrode process even in the high glucose concentration region and further in the high hematocrit value region, as is apparent from FIG. 3.

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US07/097,624 1986-09-16 1987-09-16 Whole blood diluting solution Abandoned USH602H (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP61217634A JPS6371653A (ja) 1986-09-16 1986-09-16 全血希釈液
JP61-217634 1986-09-16

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USH602H true USH602H (en) 1989-03-07

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5308767A (en) * 1986-10-31 1994-05-03 Fuji Photo Film Co., Ltd. Method for control or calibration in a chemical analytical determination
US5547874A (en) * 1986-10-31 1996-08-20 Fuji Photo Film Co., Ltd. Method for control or calibration in a chemical analytical determination
US5633169A (en) * 1995-10-27 1997-05-27 Nova Biomedical Corporation Measurement of carbon dioxide in blood
US5866349A (en) * 1989-04-25 1999-02-02 Lilja; Jan Evert Method for determination of glucose in whole blood and cuvette and photometer for carrying out said method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5308767A (en) * 1986-10-31 1994-05-03 Fuji Photo Film Co., Ltd. Method for control or calibration in a chemical analytical determination
US5547874A (en) * 1986-10-31 1996-08-20 Fuji Photo Film Co., Ltd. Method for control or calibration in a chemical analytical determination
US5866349A (en) * 1989-04-25 1999-02-02 Lilja; Jan Evert Method for determination of glucose in whole blood and cuvette and photometer for carrying out said method
US5633169A (en) * 1995-10-27 1997-05-27 Nova Biomedical Corporation Measurement of carbon dioxide in blood

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Publication number Publication date
JPS6371653A (ja) 1988-04-01

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Owner name: FUJI PHOTO FILM CO., LTD., NO. 210, NAKANUMA, MINA

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Effective date: 19880926

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