WO1981001245A1 - Methods to determine a diagnostic indicator of blood sugar conditions,and,liquid chromatographic columns therefor(cyanide free) - Google Patents

Methods to determine a diagnostic indicator of blood sugar conditions,and,liquid chromatographic columns therefor(cyanide free) Download PDF

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Publication number
WO1981001245A1
WO1981001245A1 PCT/US1980/001411 US8001411W WO8101245A1 WO 1981001245 A1 WO1981001245 A1 WO 1981001245A1 US 8001411 W US8001411 W US 8001411W WO 8101245 A1 WO8101245 A1 WO 8101245A1
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WIPO (PCT)
Prior art keywords
column bed
particles
suspension
equilibrated
test sample
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Application number
PCT/US1980/001411
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French (fr)
Inventor
K Acuff
M Volk
M Rosenthal
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Isolab Inc
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Publication date
Application filed by Isolab Inc filed Critical Isolab Inc
Priority to DE19803049920 priority Critical patent/DE3049920A1/en
Publication of WO1981001245A1 publication Critical patent/WO1981001245A1/en
Priority to JP81500132A priority patent/JPS6236181B2/ja

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Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M50/00—Constructional details or processes of manufacture of the non-active parts of electrochemical cells other than fuel cells, e.g. hybrid cells
    • H01M50/30—Arrangements for facilitating escape of gases
    • H01M50/342—Non-re-sealable arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M50/00—Constructional details or processes of manufacture of the non-active parts of electrochemical cells other than fuel cells, e.g. hybrid cells
    • H01M50/10—Primary casings, jackets or wrappings of a single cell or a single battery
    • H01M50/172—Arrangements of electric connectors penetrating the casing
    • H01M50/174—Arrangements of electric connectors penetrating the casing adapted for the shape of the cells
    • H01M50/182—Arrangements of electric connectors penetrating the casing adapted for the shape of the cells for cells with a collector centrally disposed in the active mass, e.g. Leclanché cells
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M50/00—Constructional details or processes of manufacture of the non-active parts of electrochemical cells other than fuel cells, e.g. hybrid cells
    • H01M50/30—Arrangements for facilitating escape of gases
    • H01M50/375—Vent means sensitive to or responsive to temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/10—Energy storage using batteries

Definitions

  • the invention relates to methods to de ⁇ termine a numerical percentage value as a diagnos ⁇ tic indicator of the blood sugar condition of a specific person.
  • the invention also relates to im ⁇ proved liquid chromatograp ic columns having no cyanide therein for practice of the methods.
  • a whole blood sample is collected from the person or patient and thereafter prepared as a red blood- cell hemolysate test sample using alternative clinical chemistry techniques and procedures.
  • the inven ⁇ tion provides a series of steps for separating, detecting, and measuring the amount of a group of hemoglobin species present in the test sample us ⁇ ing improved ion exchange liquid column microchroma- tographic apparatus, techniques and procedures, spectrometric analysis, and mathematical computa ⁇ tions.
  • hemoglobins are the am- photeric protein molecule coloring matter of the red blood corpuscles serving to convey oxygen to the tissues.
  • Several chromatographically separable minor hemoglobins are present in red blood cell hemolysates of normal persons.
  • Some minor hemo ⁇ globins are designated as Hb-A, Hb-A,, Hb-A, Hb-A,id,, and Hb-A,le.
  • OMPI is most prominent and accounts for the major por ⁇ tion of the minor hemoglobins. It is known that the level of hemoglobin Hb-A, has been related to a patient's average blood sugar level. Normal per- sons are expected to have 3-67 0 Hb-Al,c relative to their total hemoglobin. Untreated diabetics may have 6-12% Hb-A, relative to their total hemoglo ⁇ bin, whether the affliction is of the juvenile- onset or adult-onset type. Still further, it is understood that the levels of the species Hb-A, , as a separate and identifiable sub-group, may serve as an indicator of the degree of hypergly- cemia, an excess of sugar in the blood, over a pro ⁇ longed period of time. The assignee of the present invention,
  • the first three referenced Isolab-Acuff prior art patents ('855, '856 and '857) use an ion exchange column comprising an equilibrated suspen ⁇ sion of cellulose particles.
  • the cellulose parti ⁇ cles are of a weak base and anion exchange type in '855; of a weak acid and cation exchange type in '856 and '857.
  • the last two referenced Isolab- Acuff prior art patents (“858 and '147) use an ion exchange column of an equilibrated suspension of resin particles.
  • the resin particles are of a weak acid and cation exchange type.
  • treatment solution 28 used to prepare the ion exchange particles 27 in the form of an equilibrated suspension 27(S).
  • the treatment solution 28 suitably adjusted for pH and/or ionic strength, is also used as the "elution solution” 28 or 128, added to the column 20 after introduction of the test sample 10.
  • Each prior treatment solution 28 and elution solution 28 or 128 has been characterized by the presence of a cyanide compound or anion CN .
  • cyanide (KCN) as an ingred ⁇ ient or active compound in buffers or developers for hemoglobin chromatograms has long been accepted as essential; de riguer.
  • GMPI hemoglobin species Hb-A
  • a cyanide compound or radical in either a particle treatment solution or a buffer or elution solution. Indeed, the presence of cyanide may be considered as deleterious .
  • each of the Iso ⁇ lab-Acuff patents discloses that the amount -of hemo ⁇ globin species in a particular eluate fraction, particularly Hb-A,3.TM*C - is detected and measured by spectrometric (color) analysis.
  • Each patent spe ⁇ cifically discloses that the spectrometric analysis may be performed by an apparatus 40 which measures absorption of light caused by the hemoglobin spe ⁇ cies present in the test sample 10.
  • the prior patents rely upon the known fact that the visible portion of the spectrum for detecting the presence of a hemoglobin is in the biolet range, more speci- o fically, at substantially 415 nm or 4150 A.
  • A,.La—c having no cyanide therein but having prede- termined microchromatographic characteristics and properties for practice of the methods according to the invention.
  • the methods according to the invention to determine a numerical percentage value as a diagnostic indicator of the blood sugar condi ⁇ tion of a specific person use a whole blood sample taken from such person and thereafter prepared as a test sample containing a red blood cell hemoly- sate solution. Thereafter, a quantity of the test sample is introduced into an end of a column bed having no cyanide therein which will absorb hemo ⁇ globin species present in said test sample.
  • the column bed comprises an equilibrated suspension of ion exchange material particles having a size less than 100 mesh.
  • the particles in a particular column bed are one member selected from the class consisting of CarXH and CarYOH.
  • Car represents an inert substrate for carrying ionizable groups X providing dissociated cations H and ionizable groups Y providing disassociated anions OH .
  • CarXH particles be weakly acidic cation ex- changers and have a reported pK : 3-7 and be used in an equilibrated suspension at a pH: 6.0-75 at 22.5°C.
  • CarYOH particles be weakly basic anion exchangers and have a reported pK cL: 7-10 and be used in an equilibrated suspension at a pH: 7.3- 9.0 at 22.5°C.
  • a quantity of a buffer solu- tion having no cyanide therein is introduced into an end of the column bed to preferentially elute therefrom a first fraction which contains certain of the hemoglobin species present in the test sample.
  • the first fraction will contain substantially all of the hemoglobin species Hb-A, present in the test sample.
  • the column bed is an equilibrated suspension of CarYOH particles the first fraction will contain essentially all of the hemoglobin species present in the test sample other than, to the exclusion of, Hb-A.. In either event, an aliquot quantity of a first eluate fraction is collected from the other end of the column bed.
  • a quantity of a wash solution is intro ⁇ quizzed into an end of the column bed to desorb and elute therefrom a second fraction which will con ⁇ tain substantially all of the remaining hemoglobin species present in the test sample.
  • the second fraction will " contain essentially all of the hemoglobin species present in the test sample other than, to the exclusion of, Hb-A, .
  • the column bed is an equilibrated suspension of
  • the second fraction will contain substantially all of the hemoglobin species Hb-A, present in the test sample. In either event, an aliquot quantity of a second eluate fraction is collected from the other end of the column bed.
  • hemoglobin species present in the first and second eluate fractions are separately detected and measured by spectrometric analysis (color) and the respective amounts thereof are ex- pressed as numerical values which are then compared in accordance with a mathematical formula.
  • an aliquot quantity of a first eluate fraction is collected from an end of the column bed.
  • a quantity of a test sample is significantly diluted to provide a red blood cell hemolysate solution which may be conveniently detected and measured by spectrometric analysis.
  • the hemoglobin species present in the first eluate fraction and in the diluted hemolysate solu ⁇ tion are separately detected and measured by the spectrometric analysis (color) and the respective amounts thereof are expressed as numerical values which are then compared in accordance with a mathe ⁇ matical formula.
  • an appro ⁇ priate mathematical formula will provide a numeri- cal percentage value for the hemoglobin species Hb-A, _ present in a particular test sample for use as a diagnostic indicator of the blood sugar characteristics of the person providing that test sample.
  • the resultant numerical percentage value is available for use by qualified, specialized and medically trained personnel as a diagnostic indicator of the blood sugar characteristics of the patient provid- ing the test sample.
  • a microcolumn for use in the practice of either embodiment of the invention has a reservoir discharging into a barrel terminating in a dis ⁇ charge tip.
  • the junctures between the reservoir and barrel and the barrel and discharge tip are closed by transverse discs.
  • Ion exchange parti ⁇ cles positioned in the barrel between the discs provide a column bed.
  • the discs are permeable to a red blood cell hemolysate solution.
  • the column bed comprises an equilibrated suspension of parti-
  • - ⁇ TREAT ⁇ cles having no cyanide therein.
  • the particles will have a size of less than 100 mesh and in a particu ⁇ lar column will have one member selected from the class consisting of CarXH and CarYOH.
  • the drawing schematically shows practice of the invention specifically as to the first method embodiment wherein first and second fractions of hemoglobin species are eluted from an improved chromatographic microcolumn shown substantially in full scale.
  • Practice of the method according to the invention requires the collection of a whole blood sample from a person or patient.
  • the whole blood sample may be taken using conventional clinical chemistry techniques and procedures.
  • a procedure 10-a is used to prepare a test sample 10 which is predominantly the hemo- globin content of the whole blood sample.
  • a pro ⁇ cedure 10-b is used to prepare a test sample 10 which may include the plasma proteins, lipids, and the white and red blood cell debris, in addition to the hemoglobin content of the whole blood sample.
  • a chromatographic microcolumn is indicated generally by the numeral 20.
  • a column 20 comprises a reservoir 21 discharg ⁇ ing into a barrel 22 terminating in a discharge tip 23 selectively closed by a cap 24.
  • the juncture or intersection between the reservoir 21 and the barrel 22 is closed by a transverse plate or disc 25.
  • the juncture between the barrel 22 and the dis ⁇ charge tip 23 is also closed by a transverse plate or disc 26.
  • the ion exchange material particles comprising the column bed between the discs 25 and 26 are referred to generally by the numeral 27.
  • Each retaining disc 25 and 26 is permea ⁇ ble, having a network of micropores permitting introduction of a red blood cell hemolysate solu ⁇ tion from reservoir 21 into the barrel 22, and re ⁇ moval of an eluate fraction from " the barrel 22 through the tip 23, while retaining the column bed of particles 27 within the barrel 22.
  • the discs 25 and 26 may be made from a conventional flexible, resilient, linear, high density polyethylene of the Ziegler type. Commercially, this type of filter grade polyethylene is produced and sold under the name of Vyon.
  • the ion ex ⁇ change material particles 27 have a size less than 100 mesh and are selected from the class consisting of CarXH and CarYOH.
  • Car represents an inert substrate and may be cellulosic or a resin copoly- mer of polystyrene or methacrylic acid and divinyl- benzene.
  • X represents an ionizable group provid ⁇ ing dissociated cations H carried by a “Car” and may be a carboxyl group or a carboxymethyl group.
  • Y represents an ionizable group providing disso- ciated anions OH carried by a "Car” and may be a diethylaminoethy1 group or a mixture of amine groups having the general formulae - NH ⁇ , NER and N(R) worship.
  • the CarXH particles 27 may also be charac ⁇ terized as weakly acidic cation exchangers having a reported pK a.: 3-7.
  • the CarYOH particles 27 may also be characterized as weakly basic anion ex ⁇ changers having a reported pK :7-10.
  • the commercially available form of ion exchange material particles 27 will usually require preparation or treatment for use in a barrel 22 of a microcolumn 20 between the discs 25 and 26. Such treatment could be performed with the particles 27 in situ in the column barrel 22. However, it is preferred that the particles 27 for a series of identical columns 20 be treated using a batch technique, which will permit the use of columns 20 having predetermined microchromatographic charac ⁇ teristics and properties.
  • the ion exchange material particles 27 constituting the column bed of a column 20 are used in the form of an equilibrated suspension 27(S) having a predetermined or "starting" pH.
  • the CarXH particles are used in a suspension 27(S) at a pH: 6.0-7.5 at 22.5°C.
  • the CarYOH particles are used in a suspension 27(S) at a pH: 7.3-9.0 at 22.5°C.
  • a suspension 27(S) is prepared using a treatment solution 28, having no cyanide therein.
  • a treatment solution 28 may .also be used as buffer solution 128 for elution from a column bed, comprising a sus ⁇ pension 27(S) of either CarXH or CarYOH particles, of an eluate fraction containing certain, but not all, of the hemoglobin species present in the test sample 10.
  • a quantity of a test sample 10 prepared by either procedure 10-a or 10-b is introduced into one end of a column 20 having a column bed com ⁇ prising an equilibrated suspension 27(S) .
  • a test sample prepared according to procedure 10-a will require a 1:4 dilution using distilled water.
  • the column 20 is positioned vertically, the discharge tip cap 24 is removed and a predetermined volume of a test sample 10 is dis ⁇ charged or placed into the reservoir 21.
  • a major portion of the test sample 10 will pass readily through the disc 25 and onto the column bed of the suspension 27(S).
  • the minor portion of the test sample 10 remaining on or in the disc 25 should be purged or displaced onto the column bed using a small volume (e.g., 0.2 ml) of a solution intended for use as a buffer solution 128 in the next sequen ⁇ tial method step according to the invention.
  • a predetermined or aliquot quantity of a buffer solution 128 having no cyanide therein is dis- charged into the column reservoir 21 to preferential ⁇ ly elute from the discharge tip 23 a first fraction 30 of the test sample 10.
  • the first fraction 30 is collected in a receiver 31.
  • the column bed is a suspension 27(S) of CarXH particles
  • the first fraction 30 will contain substantially all of the hemoglobin species Hb-A, ⁇ 3.—C present in the test sample 10.
  • the column bed is a suspension 27(S) of CarYOH particles
  • the first fraction 30 will con ⁇ tain essentially all of the hemoglobin species pre- sent in the test sample 10 other than, to the ex ⁇ clusion of, Hb-A, .
  • an aliquot quantity of a first fraction 30 will be collected in a receiver 31.
  • the second fraction 32 will contain substantially all of the remaining hemoglobin species present in the test sample 10.
  • the column bed is a suspension 27(S) of CarXH particles
  • the second fraction 32 will contain essentially all of the hemoglobin spe- cies present in the test sample 10 other than Hb-
  • the second fraction 32 will con ⁇ tain substantially all of the hemoglobin species Hb-
  • A,la-c present in the test sample 10. In either event, an aliquot quantity of a second fraction 32 will be collected in a receiver 33.
  • wash solution 34 will have no cyanide therein. Otherwise, the precise formula of a wash solution 34 is not critical, so long as use thereof will not alter or modify the spectrometric absorp ⁇ tion characteristics ("color") of an elute fraction 32.
  • a compatible wash solution 34 will have either ionic strength or relative pH sufficient to fully or completely desorb substantially all the remain- ing blood components of a test sample 10 from a column bed of ion exchange material particles 27. For example, a four milliliter (4 ml) volume of 4M NaCl may be discharged into a column reservoir 21. After a period of time (e.g., 20-30 minutes), an eluate fraction of substantially 4 ml volume will be collected in a receiver 33.
  • a fraction 32 eluted from a suspension 27(S) of CarXH particles will require of suitable dilution using distilled water prior to the next sequential method step according to the invention.
  • the determination of the ratio of Hb-A, to the total hemoglobins (Hb) present in the whole blood sample collected from the person or patient uses spectrometric apparatus referred to generally by the numeral 40, following performance of liquid column microchromatographic techniques and procedures using a test sample 10.
  • the spectrometric analysis is performed by an apparatus 40 which measures absorption of light caused by the hemoglobin species present in the test sample 10. It is known that the visible portion of the spectrum for detecting the presence of a hemoglobin is in the violet range, more specifically, at substantially 415 nm or 4150 A.-
  • the apparatus 40 may be an optical spec ⁇ trometer "dedicated" or pre-set at the selected wave length of 415 nm.
  • the apparatus 40 may also be a spectrophotometer, a form of spectrometer with associated equipment which supplies the ratio, or a function of the ratio, of the radiant power of two beams as a function of an adjustably selec ⁇ ted spectral wave length. Because the spectrome ⁇ tric analysis according to the invention is for the purpose of detecting and measuring hemoglobin species from the test sample 10 by light absorp ⁇ tion characteristics, alternative forms of appara ⁇ tus 40 could be used; for example, visual compara ⁇ tors such as a set of Nessler tubes.
  • the contents of the receivers 31 and 33 are individually transferred into appropriate cu ⁇ vettes for the spectrometric apparatus 40.
  • Spec ⁇ trometric analysis of the first and second eluate fractions 30 and 32 will provide integers or natural numbers which will express, represent or indicate the amounts of hemoglobin species present in the test sample 10.
  • the displayed interger is a function of the absorbance (A) , a measurement of the amount of light of the spectral wave length of 415 nm absorbed by the hemoglobin species during passage through the cuvette and toward the sensing photocell.
  • a column bed comprising a suspension 27(S) of either CarXH or CarYOH particles and a buffer solution 128, analiquot quantity of a first eluate fraction 30 is collected in a receiver 31.
  • a quantity of the test sample 10 is prepared as a red blood cell hemolysate solution 42 which may be conveniently detected and measured by spectrometric analysis. It will' be apparent that the light absorption characteristics of the test sample 10, without significant dilution, would be of such magnitude as to impair operation efficiency of the sensing photocell of conventional spectro ⁇ metric apparatus 40.
  • a quantity of test sample 10 equal to the volume of test sample 10 introduced into an end of a column 20, prior to elution of the first fraction 30, should be diluted using distilled water in the ratio substantially 1:480 to prepare a hemolysate solution 42 for analysis by a spectrometric appara ⁇ tus 40.
  • the expressed numerical values for the separately detected and measured hemoglobin species in the eluate fraction 30 and in the diluted hemo- lysate solution 42 of a particular test sample 10 are then compared in accordance with a mathemati ⁇ cal formula.
  • a CarYOH ion exchange material particle 27 may be of the type wherein "Car”is cellulosic and Y. is a diethylaminoethyl group, -O-C ⁇ -CH ⁇ - (C ⁇ Hc) > having a reported pK of substantially 9.5.
  • this type of ion exchange ma ⁇ terial is sold under the same Whatman DEAE-52.
  • These cellulose particles 27 are pre ⁇ pared as a* suspension 27(S) having a pH of sub- stantially 8.5 at 22.5°C by mixing with a "tris" treatment solution 28 of the formula 6.06g ⁇ N.C
  • a quantity of a buffer solution 128 is introduced into an end of a column bed of a sus ⁇ pension 27(S)of these cellulose particles to pre ⁇ ferentially elute therefrom an eluate fraction 30 containing essentially of the hemoglobin species present in a test sample 10 other than Hb-A-, .
  • the buffer solution 128 may be a "tris" solution of the same formula as the treatment solution 28 with the pH adjusted to substantially 7.7 at 22.5°C using concentrated HCl.
  • Example 2 Another CarYOH ion exchange material particle 27 may be of the type wherein "Car” is a resin copolymer of polystyrene and divinylbenzene and Y is a mixture of primary, secondary, and ter ⁇ tiary amine groups having the general formulae - H «, NHR and N(R) 2. where R is an aliphatic hydro ⁇ carbon radical such as -CE- or -C- c - These par ⁇ ticles 27 have a reported pK : 7-9. Commercially, this type of ion exchange material is sold under the names Dowdex MWA-1 and Amberlite IRA-93.
  • resin particles 27 may be pre ⁇ pared as a suspension 27(S) having a pH of sub ⁇ stantially 7.5 at 22.5°C by mixing with a "tris" treatment solution 28 of the same formula as Exam ⁇ ple 1.
  • a column bed of a suspension 27(S) of these resin particles may be eluted from a column bed of a suspension 27(S) of these resin particles by use of a "tris" buffer solution 128 of the same formula as in Example 1, with a pH ad ⁇ justed to substantially 7.5 at 22.5°C.
  • a CarXH ion exchange material parti ⁇ cle 27 may be of the type wherein "Car” is a resin copolymer of methacrylic acid and divinylbenzene and X is a carboxyl group, -C00H, having a reported pK 3.: 4-6.
  • this type of ion ex- change material is sold under the name Amberlite CG-50.
  • the resin polymers 27 are prepared as a suspension 27(S) having a pH of substantialy 6.98 at 22.5°C by mixing with "phosphate" treatment solution 28 of the formula: 3.74g KH 2 PO ⁇ (0.027M), 0.955g KOH (0.017 M) , with 0.10g NaN 3 (0.01%) as a preservative, made up in one liter of H ⁇ O. After mixing with the treatment solution 28, the resin particles 727 are then further treated with an acid solution 29 (e.g., 4 M H PO,) to readjust to the pH of substantially 6.98.
  • "phosphate" treatment solution 28 of the formula: 3.74g KH 2 PO ⁇ (0.027M), 0.955g KOH (0.017 M) , with 0.10g NaN 3 (0.01%) as a preservative, made up in one liter of H ⁇ O.
  • an acid solution 29 e.g., 4 M H PO,
  • a quantity of a buffer solution 128 is intro ⁇ quizzed into an end of a column bed of a suspension 27(S) of these resin particles to preferentially elute therefrom an eluate fraction 30 containing substantially all of the hemoglobin species Hb- A-, present in a test sample 10.
  • the buffer so ⁇ lution 128 may be a "phosphate" solution of the same formula as the treatment solution 28 and hav- ing a pH of substantially 6.98.
  • CarXH ion exchange material particle 27 may be of the type wherein "Car” is cellulosic and X ⁇ is a carboxymethyl group, -0- CH 2 ⁇ C00H, having a reported pK of 3.5. Commer ⁇ cially, this type of ion exchange material is sold under the name Whatman CM-52.
  • These cellulose particles 27 may be prepared as a suspension 27(S) having a pH of sub ⁇ stantially 6.8 at 22.5°C by mixing with "phosphate" treatment solution 28 of the formula: 3.74g KH 2 P0, (0.027 M) , and 0.748g KOH (0.013 M) , with 0.10g NaN (0.01%) -as a preservative, made up in one liter of H 2 0.
  • "phosphate" treatment solution 28 of the formula: 3.74g KH 2 P0, (0.027 M) , and 0.748g KOH (0.013 M) , with 0.10g NaN (0.01%) -as a preservative, made up in one liter of H 2 0.
  • a first fraction 30 of a test sample 10, containing substantially all of the hemoglobin species Hb-A, may be eluted from a column bed of a suspension 27(S) of these cellulose particles by use of a "phosphate" buffer solution 128 of the same formula as the treatment solution
  • Example 5 The cellulose particles 27 of Example 4 may be prepared as a suspension 27(S) having a pH of substantially 6.1 at 22.5°C by mixing with a "bis-tris" treatment solution 28 of the formula: 6.28g (HOCH 2 CH 2 ) 2 NC (CH 2 OH 3 (0.03M), with 0.10g N (0.01%) as a preservative, made up in one liter of H 2 0. After mixing with the treatment solution 28, the cellulose particles 27 are then further treated with an acid solution 29 (e.g. , 4 M HC1) to readjust to the pH of substantially 6.1. A first fraction 30 of a test sample
  • a "bis-tris" treatment solution 28 of the formula: 6.28g (HOCH 2 CH 2 ) 2 NC (CH 2 OH 3 (0.03M), with 0.10g N (0.01%) as a preservative, made up in one liter of H 2 0.
  • an acid solution 29 e.g. , 4 M HC1
  • a column bed of a suspension 27(S) of these cellulose particles may be eluted from a column bed of a suspension 27(S) of these cellulose particles by the use of a "bis-tris" buffer solution 128 of the same formula as the treatment solution 28 with the addition of 2.34g NaCl (0.04 M) and having a pH of substantially 6.1.
  • Example 6 The resin particles of Example 3 may be prepared as a suspension 27(S) having a pH of substantially 6.8 at 22.5°C by mixing with a "bis- tris" treatment solution 28, of the same formula as in Example 5.
  • a first fraction 30 of a test sample 10, containing substantially all of the hemoglobin species Hb-A,la—c, may be eluted from a column bed of a suspension 27(S) of these resin particles by the use of a "bis-tris" buffer solu ⁇ tion 128 of the same formula as the treatment solu ⁇ tion 28 with the addition of 7.02g NaCl (0.12M), having a pH of substantially 6.8.

Abstract

Methods and microcolumns (20) to determine a numerical percentage value as a diagnostic indicator of the blood sugar condition of a specific person. A whole blood sample (10) is lysed and a test sample thereof is placed on a microchromatographic column bed of ion exchange material particles (27). The column bed is characterized by having no cyanide therein. The column bed comprises an equilibrated suspension of particles selected from the class consisting of CarXH and CarYOH, where "Car" represents an inert substrate for carrying ionizable groups X- providing dissociated cations H+ and ionizable groups Y+ providing anions OH-. Fractions (31, 32) of various hemoglobin species in the test sample are preferentially eluted or desorbed from the column bed by buffer and wash solutions characterized by having no cyanide therein. Amounts of hemoglobin species in the eluate fractions are detected and measured by spectrometric (color) analysis (40). A mathematical computation using integer factors corresponding to amounts of various hemoglobin species produces the numerical percentage value.

Description

1
METHODS TO DETERMINE A DIAGNOSTIC INDICATOR OF BLOOD SUGAR CONDITIONS, AND, LIQUID CHROMATOGRAPHIC COLUMNS THEREFOR (CYANIDE FREE)
TECHNICAL FIELD
The invention relates to methods to de¬ termine a numerical percentage value as a diagnos¬ tic indicator of the blood sugar condition of a specific person. The invention also relates to im¬ proved liquid chromatograp ic columns having no cyanide therein for practice of the methods.
BACKGROUND ART
According to the invention, a whole blood sample is collected from the person or patient and thereafter prepared as a red blood- cell hemolysate test sample using alternative clinical chemistry techniques and procedures. Thereafter, the inven¬ tion provides a series of steps for separating, detecting, and measuring the amount of a group of hemoglobin species present in the test sample us¬ ing improved ion exchange liquid column microchroma- tographic apparatus, techniques and procedures, spectrometric analysis, and mathematical computa¬ tions.
In biochemistry, hemoglobins are the am- photeric protein molecule coloring matter of the red blood corpuscles serving to convey oxygen to the tissues. Several chromatographically separable minor hemoglobins are present in red blood cell hemolysates of normal persons. Some minor hemo¬ globins are designated as Hb-A, Hb-A,, Hb-A, Hb-A,id,, and Hb-A,le. The hemog&lobin species Hb-A, ιc
OMPI is most prominent and accounts for the major por¬ tion of the minor hemoglobins. It is known that the level of hemoglobin Hb-A, has been related to a patient's average blood sugar level. Normal per- sons are expected to have 3-670 Hb-Al,c relative to their total hemoglobin. Untreated diabetics may have 6-12% Hb-A, relative to their total hemoglo¬ bin, whether the affliction is of the juvenile- onset or adult-onset type. Still further, it is understood that the levels of the species Hb-A, , as a separate and identifiable sub-group, may serve as an indicator of the degree of hypergly- cemia, an excess of sugar in the blood, over a pro¬ longed period of time. The assignee of the present invention,
Isolab, Incorporated, is now the owner of five United States patents relating to methods and columns similar to the methods and columns disclosed herein. These patents are: No. 4,142,855; No. 4, 142,856; No. 4,142,857; and No. 4,142,858, each granted March/1979 to Acuff; and No. 4,168,147, September/1979, also to Acuff.
The first three referenced Isolab-Acuff prior art patents ('855, '856 and '857) use an ion exchange column comprising an equilibrated suspen¬ sion of cellulose particles. The cellulose parti¬ cles are of a weak base and anion exchange type in '855; of a weak acid and cation exchange type in '856 and '857. The last two referenced Isolab- Acuff prior art patents ("858 and '147) use an ion exchange column of an equilibrated suspension of resin particles. The resin particles are of a weak acid and cation exchange type.
In each of the Isolab-Acuff patents, reference is made to a "treatment solution" 28 used to prepare the ion exchange particles 27 in the form of an equilibrated suspension 27(S). In each patent, the treatment solution 28, suitably adjusted for pH and/or ionic strength, is also used as the "elution solution" 28 or 128, added to the column 20 after introduction of the test sample 10. Each prior treatment solution 28 and elution solution 28 or 128 has been characterized by the presence of a cyanide compound or anion CN . The use of cyanide (KCN) as an ingred¬ ient or active compound in buffers or developers for hemoglobin chromatograms has long been accepted as essential; de riguer. In the prior literature, a paper by Allen et al., Observations on the Chroma- tographic Heterogeneity of Normal Adult and Fetal Human Hemoglobin: A Study of the Effects and Cry¬ stallization and Chromatography on the Heterogenei¬ ty and Isoleucine Content, Journal of the American Chemical Society, Vol. 80, pp. 1628-1634, April 1958, discloses that "[pjotassium cyanide was ori¬ ginally included in the developers in order to de¬ crease the dissociation of ferrihemoglobin cyanide during chromatography. It was not removed from the developers when oxyhemoglobin was chromato- graphed because ferrihemoglobin cyanide and oxy¬ hemoglobin have identical chromatographic behavior. Thus, traces of ferrihemoglobin (methemoglobin) in solutions of oxyhemoglobin are converted to ferrihemoglobin cyanide and do not produce slow moving extraneous zones on the column." supra, p. 1630.
Subsequent to the inventions described in the Isolab-Acuff patents, it has been discovered that microchromatographic clinical techniques and procedures to determine an indication or level of
GMPI the hemoglobin species Hb-A, do not require the presence of' a cyanide compound or radical, in either a particle treatment solution or a buffer or elution solution. Indeed, the presence of cyanide may be considered as deleterious .
By way of illustration, each of the Iso¬ lab-Acuff patents discloses that the amount -of hemo¬ globin species in a particular eluate fraction, particularly Hb-A,3.™*C - is detected and measured by spectrometric (color) analysis. Each patent spe¬ cifically discloses that the spectrometric analysis may be performed by an apparatus 40 which measures absorption of light caused by the hemoglobin spe¬ cies present in the test sample 10. The prior patents rely upon the known fact that the visible portion of the spectrum for detecting the presence of a hemoglobin is in the biolet range, more speci- o fically, at substantially 415 nm or 4150 A.
After filing of the applications pre- ceding the Isolab-Acuff patents, it became apparent that the integers expressing the amounts of hemo¬ globin species present in each eluate fraction (as determined by spectrometric analysis at 415 nm) were affected by a factor of time. When the spec- trometric analysis was performed rather promptly (e.g., 30 minutes after elution), the resultant integer would conform to a norm or standard. How¬ ever, after a longer period of time (e.g., 60 minutes) , the resultant integer would reflect a lower value for the hemoglobin species Hb-A, After careful analysis of all aspects of the methods and columns disclosed in the Isolab-Acuff patents, it has now been determined, and is there¬ fore specified as being critical, that the presence of a cyanide compound or radical contributed to
. an inaccuracy in the true or reasonably correct in¬ teger reflecting the hemoglobin species Hb-A, present in a particular and specific test sample. It is now understood that the presence of cyanide in either the ion exchange particle treatment solution or the buffer solution will in¬ crease the incidence of a time-factored conversion of ferrous hemoglobin derivatives to ferrihemo¬ globin cyanide, with an attendant alteration in the integer or value obtained by spectrometric analysis. Of course, a skilled laboratory person or techni¬ cian would (and will) compensate for such varia¬ tion. However, provision of a standard test that can (and will) be performed by relatively unskilled personnel mandates the use of a technique and pro¬ cedure which does not employ cyanide.
Other advantage's of non-use of cyanide will include the facilitation of export-import of microcolumns and reagents according to the inven- tion. In many countries throughout the world, labeling of a product as containing cyanide (even in trace amounts) presents regulatory complications. In countries of product manufacture, elimination of wastes or residue relating to either reagent pre- paration or ion exchange material equilibration can (and will) give cause to environmental hazard situations and legitimate governmental entity con¬ cern. Finally, disposal of used columns and reagent solutions is simplified.
DISCLOSURE OF INVENTION
It is an object of the invention to pro¬ vide methods to determine a numerical percentage value as a diagnostic indicator of the blood sugar
MPI condition of a specific person, without the use of reagents or solutions containing any amounts, trace or otherwise, of any cyanide compound or anion CN .
It is a further object to provide a method which will separate, detect and measure the ratio of the sub-gro !up of hemoglobin species Hb-A,ιa—c to the total hemoglobins (Hb) present in the blood of a specific person quickly, inexpensively, accur¬ ately and without the presence of a cyanide co - pound or the anion CN .
It is still further an object to provide a method which, although requiring of a number of sequential or consecutive steps, is of such a charac¬ ter and nature as to permit the adoption of pro- cedures and protocols which may become standard and routine, permitting persons skilled in the art of clinical chemistry to repeatedly and accurately test the blood of large groups of persons to estab¬ lish a data base for use by qualified, specialized and medically trained personnel in diagnosing the blood sugar condition of specific persons suspected as being diabetic.
It is still another object of the inven¬ tion to provide microcolumns, for chromatographic clinical techniques and procedures to determine an indication or level of the hemoglobin species Hb-
A,.La—c, having no cyanide therein but having prede- termined microchromatographic characteristics and properties for practice of the methods according to the invention.
These and other objects of the invention, as well as the advantages thereof, will be apparent in view of the detailed descriptions of the various embodiments thereof as set forth below. In general, the methods according to the invention to determine a numerical percentage value as a diagnostic indicator of the blood sugar condi¬ tion of a specific person use a whole blood sample taken from such person and thereafter prepared as a test sample containing a red blood cell hemoly- sate solution. Thereafter, a quantity of the test sample is introduced into an end of a column bed having no cyanide therein which will absorb hemo¬ globin species present in said test sample. The column bed comprises an equilibrated suspension of ion exchange material particles having a size less than 100 mesh. The particles in a particular column bed are one member selected from the class consisting of CarXH and CarYOH. "Car" represents an inert substrate for carrying ionizable groups X providing dissociated cations H and ionizable groups Y providing disassociated anions OH .
It has been determined as critical and essential for the practice of the invention that the CarXH particles be weakly acidic cation ex- changers and have a reported pK : 3-7 and be used in an equilibrated suspension at a pH: 6.0-75 at 22.5°C.
It has also been determined as critical and essential for the practice of the invention that the CarYOH particles be weakly basic anion exchangers and have a reported pK cL: 7-10 and be used in an equilibrated suspension at a pH: 7.3- 9.0 at 22.5°C.
Thereafter a quantity of a buffer solu- tion having no cyanide therein is introduced into an end of the column bed to preferentially elute therefrom a first fraction which contains certain of the hemoglobin species present in the test sample. When the column bed is an equilibrated suspension of CarXH particles, the first fraction will contain substantially all of the hemoglobin species Hb-A, present in the test sample. When the column bed is an equilibrated suspension of CarYOH particles the first fraction will contain essentially all of the hemoglobin species present in the test sample other than, to the exclusion of, Hb-A.. In either event, an aliquot quantity of a first eluate fraction is collected from the other end of the column bed. In the first method embodiment of the in¬ vention, a quantity of a wash solution is intro¬ duced into an end of the column bed to desorb and elute therefrom a second fraction which will con¬ tain substantially all of the remaining hemoglobin species present in the test sample. When the column bed is an equilibrated suspension of CarXH particles , the second fraction will" contain essentially all of the hemoglobin species present in the test sample other than, to the exclusion of, Hb-A, . When the column bed is an equilibrated suspension of
CarYOH particles, the second fraction will contain substantially all of the hemoglobin species Hb-A, present in the test sample. In either event, an aliquot quantity of a second eluate fraction is collected from the other end of the column bed.
Then, the hemoglobin species present in the first and second eluate fractions are separately detected and measured by spectrometric analysis (color) and the respective amounts thereof are ex- pressed as numerical values which are then compared in accordance with a mathematical formula.
In the second method embodiment of the invention, using a column bed having an equilibra¬ ted suspension of either CarXH or CarYOH particles and a buffer solution, an aliquot quantity of a first eluate fraction is collected from an end of the column bed. Also, a quantity of a test sample is significantly diluted to provide a red blood cell hemolysate solution which may be conveniently detected and measured by spectrometric analysis. Then, the hemoglobin species present in the first eluate fraction and in the diluted hemolysate solu¬ tion are separately detected and measured by the spectrometric analysis (color) and the respective amounts thereof are expressed as numerical values which are then compared in accordance with a mathe¬ matical formula.
In either method embodiment, an appro¬ priate mathematical formula will provide a numeri- cal percentage value for the hemoglobin species Hb-A, _ present in a particular test sample for use as a diagnostic indicator of the blood sugar characteristics of the person providing that test sample. In all embodiments of the invention, the resultant numerical percentage value is available for use by qualified, specialized and medically trained personnel as a diagnostic indicator of the blood sugar characteristics of the patient provid- ing the test sample.
A microcolumn for use in the practice of either embodiment of the invention has a reservoir discharging into a barrel terminating in a dis¬ charge tip. The junctures between the reservoir and barrel and the barrel and discharge tip are closed by transverse discs. Ion exchange parti¬ cles positioned in the barrel between the discs provide a column bed. The discs are permeable to a red blood cell hemolysate solution. The column bed comprises an equilibrated suspension of parti-
-^TREATΓ cles having no cyanide therein. The particles will have a size of less than 100 mesh and in a particu¬ lar column will have one member selected from the class consisting of CarXH and CarYOH.
BRIEF DESCRIPTION OF DRAWINGS
The drawing schematically shows practice of the invention specifically as to the first method embodiment wherein first and second fractions of hemoglobin species are eluted from an improved chromatographic microcolumn shown substantially in full scale.
BEST MODE FOR CARRYING OUT THE INVENTION
Practice of the method according to the invention requires the collection of a whole blood sample from a person or patient. The whole blood sample may be taken using conventional clinical chemistry techniques and procedures.
The disclosure of the Isolab-Acuff prior art patents describing in detail two general state of the art procedures for preparing a suitable test sample, referred to generally by the numeral 10, containing a red blood cell hemolysate solution from a whole blood sample, is incorporated herein by reference. A procedure 10-a is used to prepare a test sample 10 which is predominantly the hemo- globin content of the whole blood sample. A pro¬ cedure 10-b is used to prepare a test sample 10 which may include the plasma proteins, lipids, and the white and red blood cell debris, in addition to the hemoglobin content of the whole blood sample. With reference to the drawing, which is substantially in full scale, a chromatographic microcolumn is indicated generally by the numeral 20. A column 20 comprises a reservoir 21 discharg¬ ing into a barrel 22 terminating in a discharge tip 23 selectively closed by a cap 24. The juncture or intersection between the reservoir 21 and the barrel 22 is closed by a transverse plate or disc 25. The juncture between the barrel 22 and the dis¬ charge tip 23 is also closed by a transverse plate or disc 26. The ion exchange material particles comprising the column bed between the discs 25 and 26 are referred to generally by the numeral 27.
Each retaining disc 25 and 26 is permea¬ ble, having a network of micropores permitting introduction of a red blood cell hemolysate solu¬ tion from reservoir 21 into the barrel 22, and re¬ moval of an eluate fraction from "the barrel 22 through the tip 23, while retaining the column bed of particles 27 within the barrel 22. The discs 25 and 26 may be made from a conventional flexible, resilient, linear, high density polyethylene of the Ziegler type. Commercially, this type of filter grade polyethylene is produced and sold under the name of Vyon. According to the invention, the ion ex¬ change material particles 27 have a size less than 100 mesh and are selected from the class consisting of CarXH and CarYOH. "Car" represents an inert substrate and may be cellulosic or a resin copoly- mer of polystyrene or methacrylic acid and divinyl- benzene. X represents an ionizable group provid¬ ing dissociated cations H carried by a "Car" and may be a carboxyl group or a carboxymethyl group. Y represents an ionizable group providing disso- ciated anions OH carried by a "Car" and may be a diethylaminoethy1 group or a mixture of amine groups having the general formulae - NH^, NER and N(R)„.
The CarXH particles 27 may also be charac¬ terized as weakly acidic cation exchangers having a reported pK a.: 3-7. The CarYOH particles 27 may also be characterized as weakly basic anion ex¬ changers having a reported pK :7-10.
The commercially available form of ion exchange material particles 27 will usually require preparation or treatment for use in a barrel 22 of a microcolumn 20 between the discs 25 and 26. Such treatment could be performed with the particles 27 in situ in the column barrel 22. However, it is preferred that the particles 27 for a series of identical columns 20 be treated using a batch technique, which will permit the use of columns 20 having predetermined microchromatographic charac¬ teristics and properties.
The ion exchange material particles 27 constituting the column bed of a column 20 are used in the form of an equilibrated suspension 27(S) having a predetermined or "starting" pH. The CarXH particles are used in a suspension 27(S) at a pH: 6.0-7.5 at 22.5°C. The CarYOH particles are used in a suspension 27(S) at a pH: 7.3-9.0 at 22.5°C. A suspension 27(S) is prepared using a treatment solution 28, having no cyanide therein.
According to the invention, a treatment solution 28 may .also be used as buffer solution 128 for elution from a column bed, comprising a sus¬ pension 27(S) of either CarXH or CarYOH particles, of an eluate fraction containing certain, but not all, of the hemoglobin species present in the test sample 10. A quantity of a test sample 10 prepared by either procedure 10-a or 10-b is introduced into one end of a column 20 having a column bed com¬ prising an equilibrated suspension 27(S) . A test sample prepared according to procedure 10-a will require a 1:4 dilution using distilled water.
Preferably, the column 20 is positioned vertically, the discharge tip cap 24 is removed and a predetermined volume of a test sample 10 is dis¬ charged or placed into the reservoir 21. A major portion of the test sample 10 will pass readily through the disc 25 and onto the column bed of the suspension 27(S). The minor portion of the test sample 10 remaining on or in the disc 25 should be purged or displaced onto the column bed using a small volume (e.g., 0.2 ml) of a solution intended for use as a buffer solution 128 in the next sequen¬ tial method step according to the invention.
A predetermined or aliquot quantity of a buffer solution 128 having no cyanide therein is dis- charged into the column reservoir 21 to preferential¬ ly elute from the discharge tip 23 a first fraction 30 of the test sample 10. The first fraction 30 is collected in a receiver 31. When the column bed is a suspension 27(S) of CarXH particles, the first fraction 30 will contain substantially all of the hemoglobin species Hb-A, ■ 3.—C present in the test sample 10. When the column bed is a suspension 27(S) of CarYOH particles, the first fraction 30 will con¬ tain essentially all of the hemoglobin species pre- sent in the test sample 10 other than, to the ex¬ clusion of, Hb-A, . In either event, after a period of time following addition of the buffer 28, an aliquot quantity of a first fraction 30 will be collected in a receiver 31. In the first method embodiment of the in-
'~ vention, as shown in the drawing, a second fraction
32 of the test sample 10 is collected in a receiver
33 after an aliquot quantity of a wash solution 34 is discharged into the column reservoir 21. The second fraction 32 will contain substantially all of the remaining hemoglobin species present in the test sample 10. When the column bed is a suspension 27(S) of CarXH particles, the second fraction 32 will contain essentially all of the hemoglobin spe- cies present in the test sample 10 other than Hb-
A, -Lcl—C. When the column bed is a suspension 27(S) of
CarYOH particles, the second fraction 32 will con¬ tain substantially all of the hemoglobin species Hb-
A,la-c present in the test sample 10. In either event, an aliquot quantity of a second fraction 32 will be collected in a receiver 33.
The wash solution 34 will have no cyanide therein. Otherwise, the precise formula of a wash solution 34 is not critical, so long as use thereof will not alter or modify the spectrometric absorp¬ tion characteristics ("color") of an elute fraction 32. A compatible wash solution 34 will have either ionic strength or relative pH sufficient to fully or completely desorb substantially all the remain- ing blood components of a test sample 10 from a column bed of ion exchange material particles 27. For example, a four milliliter (4 ml) volume of 4M NaCl may be discharged into a column reservoir 21. After a period of time (e.g., 20-30 minutes), an eluate fraction of substantially 4 ml volume will be collected in a receiver 33. A fraction 32 eluted from a suspension 27(S) of CarXH particles will require of suitable dilution using distilled water prior to the next sequential method step according to the invention. According to both method embodiments of the invention, the determination of the ratio of Hb-A, to the total hemoglobins (Hb) present in the whole blood sample collected from the person or patient uses spectrometric apparatus referred to generally by the numeral 40, following performance of liquid column microchromatographic techniques and procedures using a test sample 10.
The spectrometric analysis is performed by an apparatus 40 which measures absorption of light caused by the hemoglobin species present in the test sample 10. It is known that the visible portion of the spectrum for detecting the presence of a hemoglobin is in the violet range, more specifically, at substantially 415 nm or 4150 A.-
The apparatus 40 may be an optical spec¬ trometer "dedicated" or pre-set at the selected wave length of 415 nm. The apparatus 40 may also be a spectrophotometer, a form of spectrometer with associated equipment which supplies the ratio, or a function of the ratio, of the radiant power of two beams as a function of an adjustably selec¬ ted spectral wave length. Because the spectrome¬ tric analysis according to the invention is for the purpose of detecting and measuring hemoglobin species from the test sample 10 by light absorp¬ tion characteristics, alternative forms of appara¬ tus 40 could be used; for example, visual compara¬ tors such as a set of Nessler tubes. In the first method embodiment of the in¬ vention, the contents of the receivers 31 and 33 are individually transferred into appropriate cu¬ vettes for the spectrometric apparatus 40. Spec¬ trometric analysis of the first and second eluate fractions 30 and 32 will provide integers or natural numbers which will express, represent or indicate the amounts of hemoglobin species present in the test sample 10. When using a conventional spectrometer or spectrophotometer as the apparatus 40, the displayed interger is a function of the absorbance (A) , a measurement of the amount of light of the spectral wave length of 415 nm absorbed by the hemoglobin species during passage through the cuvette and toward the sensing photocell. The expressed numerical values for the separately detected and measured hemoglobin species in each eluate fraction 30 and 32 of a particular test sample 10 are then compared in accordance with a mathematical formula. When the eluate fractions 30 and 32 are eluted from a column bed comprising a suspen¬ sion 27(S) of CarXH particles, the computation is
integer for first fraction 30 x 100 _ integer for first fraction 30 + integer for second fraction 32 a numerical percentage value. When the eluate fractions 30 and 32 are eluted from a column bed comprising a suspen¬ sion 27(S) of CarYOH particles, the computation is
integer for second fraction 32 x 100 _ integer for first fraction 30 + integer for second fraction 32 a numerical percentage value.
In the second embodiment of the inven¬ tion, using a column bed comprising a suspension 27(S) of either CarXH or CarYOH particles and a buffer solution 128, analiquot quantity of a first eluate fraction 30 is collected in a receiver 31. Also, either at a prior time, concurrently or con- secutively, a quantity of the test sample 10 is prepared as a red blood cell hemolysate solution 42 which may be conveniently detected and measured by spectrometric analysis. It will' be apparent that the light absorption characteristics of the test sample 10, without significant dilution, would be of such magnitude as to impair operation efficiency of the sensing photocell of conventional spectro¬ metric apparatus 40. Accordingly, and by way of example, a quantity of test sample 10 equal to the volume of test sample 10 introduced into an end of a column 20, prior to elution of the first fraction 30, should be diluted using distilled water in the ratio substantially 1:480 to prepare a hemolysate solution 42 for analysis by a spectrometric appara¬ tus 40.
The expressed numerical values for the separately detected and measured hemoglobin species in the eluate fraction 30 and in the diluted hemo- lysate solution 42 of a particular test sample 10 are then compared in accordance with a mathemati¬ cal formula.
When the eluate fraction 30 is eluted from a column bed comprising a suspension 27(S) of CarXH particles, the computation is
integer for eluate fraction 30 x 100.._ integer for hemolysate solution 42 a numerical percentage value. When the eluate fraction 30 is eluted from a column bed comprising 27(S) of CarYOH par¬ ticles, the computation is
inn - (intefier f°r eluate fraction 30 x 100) _ (integer for hemolysate solution 42) a numerical percentage value. Use any of these mathematical compu¬ tations will provide a numerical value for the hemoglobin species Hb-A., present in a particu- lar test sample 10 for use as a diagnostic indica- tor of the blood sugar characteristics of the per¬ son providing that test sample.
The following Examples will further illustrate and describe the practice of the inven¬ tion by persons skilled in the art of clinical chemistry.
Example 1 A CarYOH ion exchange material particle 27 may be of the type wherein "Car"is cellulosic and Y. is a diethylaminoethyl group, -O-C^-CH^- (C^Hc) > having a reported pK of substantially 9.5. Commercially, this type of ion exchange ma¬ terial is sold under the same Whatman DEAE-52.
These cellulose particles 27 are pre¬ pared as a* suspension 27(S) having a pH of sub- stantially 8.5 at 22.5°C by mixing with a "tris" treatment solution 28 of the formula 6.06g ^N.C
(CH20H)3 (0.05 M) , with 0.10g Na 3 (0.01%) as a preservative, made up in one liter of H- 0. After mixing with the treatment solution 28, the cellu- lose particles 27 are then further treated with an acid solution 29 (e.g., 4 M HC1) to readjust to the pH of substantially 8.5.
In both method embodiments of the in¬ vention, a quantity of a buffer solution 128 is introduced into an end of a column bed of a sus¬ pension 27(S)of these cellulose particles to pre¬ ferentially elute therefrom an eluate fraction 30 containing essentially of the hemoglobin species present in a test sample 10 other than Hb-A-, . The buffer solution 128 may be a "tris" solution of the same formula as the treatment solution 28 with the pH adjusted to substantially 7.7 at 22.5°C using concentrated HCl.
Example 2 Another CarYOH ion exchange material particle 27 may be of the type wherein "Car" is a resin copolymer of polystyrene and divinylbenzene and Y is a mixture of primary, secondary, and ter¬ tiary amine groups having the general formulae - H«, NHR and N(R)2. where R is an aliphatic hydro¬ carbon radical such as -CE- or -C- c - These par¬ ticles 27 have a reported pK : 7-9. Commercially, this type of ion exchange material is sold under the names Dowdex MWA-1 and Amberlite IRA-93. These resin particles 27 may be pre¬ pared as a suspension 27(S) having a pH of sub¬ stantially 7.5 at 22.5°C by mixing with a "tris" treatment solution 28 of the same formula as Exam¬ ple 1. A first fraction 30 of a test sample
10, containing essentially all of the hemoglobin species other than Hb-A, , may be eluted from a column bed of a suspension 27(S) of these resin particles by use of a "tris" buffer solution 128 of the same formula as in Example 1, with a pH ad¬ justed to substantially 7.5 at 22.5°C.
Example 3 A CarXH ion exchange material parti¬ cle 27 may be of the type wherein "Car" is a resin copolymer of methacrylic acid and divinylbenzene and X is a carboxyl group, -C00H, having a reported pK 3.: 4-6. Commercially, this type of ion ex- change material is sold under the name Amberlite CG-50. The resin polymers 27 are prepared as a suspension 27(S) having a pH of substantialy 6.98 at 22.5°C by mixing with "phosphate" treatment solution 28 of the formula: 3.74g KH2PO^ (0.027M), 0.955g KOH (0.017 M) , with 0.10g NaN3 (0.01%) as a preservative, made up in one liter of H^O. After mixing with the treatment solution 28, the resin particles 727 are then further treated with an acid solution 29 (e.g., 4 M H PO,) to readjust to the pH of substantially 6.98.
In both method embodiments of the inven¬ tion, a quantity of a buffer solution 128 is intro¬ duced into an end of a column bed of a suspension 27(S) of these resin particles to preferentially elute therefrom an eluate fraction 30 containing substantially all of the hemoglobin species Hb- A-, present in a test sample 10. The buffer so¬ lution 128 may be a "phosphate" solution of the same formula as the treatment solution 28 and hav- ing a pH of substantially 6.98.
Example 4
Another CarXH ion exchange material particle 27 may be of the type wherein "Car" is cellulosic and X~ is a carboxymethyl group, -0- CH2~C00H, having a reported pK of 3.5. Commer¬ cially, this type of ion exchange material is sold under the name Whatman CM-52.
These cellulose particles 27 may be prepared as a suspension 27(S) having a pH of sub¬ stantially 6.8 at 22.5°C by mixing with "phosphate" treatment solution 28 of the formula: 3.74g KH2P0, (0.027 M) , and 0.748g KOH (0.013 M) , with 0.10g NaN (0.01%) -as a preservative, made up in one liter of H20. A first fraction 30 of a test sample 10, containing substantially all of the hemoglobin species Hb-A, , may be eluted from a column bed of a suspension 27(S) of these cellulose particles by use of a "phosphate" buffer solution 128 of the same formula as the treatment solution 28 and having a pH of substantially 6.8.
Example 5 The cellulose particles 27 of Example 4 may be prepared as a suspension 27(S) having a pH of substantially 6.1 at 22.5°C by mixing with a "bis-tris" treatment solution 28 of the formula: 6.28g (HOCH2CH2)2 NC (CH2OH 3 (0.03M), with 0.10g N (0.01%) as a preservative, made up in one liter of H20. After mixing with the treatment solution 28, the cellulose particles 27 are then further treated with an acid solution 29 (e.g. , 4 M HC1) to readjust to the pH of substantially 6.1. A first fraction 30 of a test sample
10, containing substantially all of the hemoglobin species Hb-A, , may be eluted from a column bed of a suspension 27(S) of these cellulose particles by the use of a "bis-tris" buffer solution 128 of the same formula as the treatment solution 28 with the addition of 2.34g NaCl (0.04 M) and having a pH of substantially 6.1.
Example 6 The resin particles of Example 3 may be prepared as a suspension 27(S) having a pH of substantially 6.8 at 22.5°C by mixing with a "bis- tris" treatment solution 28, of the same formula as in Example 5. A first fraction 30 of a test sample 10, containing substantially all of the hemoglobin species Hb-A,la—c, may be eluted from a column bed of a suspension 27(S) of these resin particles by the use of a "bis-tris" buffer solu¬ tion 128 of the same formula as the treatment solu¬ tion 28 with the addition of 7.02g NaCl (0.12M), having a pH of substantially 6.8.
SUMMARY In all embodiments of the invention as described, several steps, techniques or proce- dures are disclosed wherein dilution, using dis¬ tilled water, is either required or suggested. It will be understood by a person skilled in the art of clinical chemistry that the best modes of prac¬ ticing the invention using an improved microcolumn 20 will require careful adoption and consistent following of routine procedures, if the invention is to represent a reliable method of assessing the presence of diabetes and monitoring the degree of diabetic control. It will be further understood by a practitioner of the invention that a procedure or protocol for repetitive testing of large numbers of persons, both diabetic and normal, will inherent¬ ly incorporate therein: standard quantities and volumes of test samples 10, solutions 28 and 128, 29 and 34, fractions 30 and 32 and solution 42. consistent and compatible dilution ratios; and, careful selection and regulation of the spectro¬ metric apparatus 40. Therefore, the full scope and extent of the invention should be determined solely by the words of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:
1. A method to determine a numerical percentage value as a diagnostic indicator of the blood sugar condition of a specific person, wherein a whole blood sample is taken from said person and thereafter prepared as a test sample containing a red blood cell hemolysate solution, and there¬ after, a quantity of said test sample is intro¬ duced into an end of a column bed having no cyanide therein which will absorb hemoglobin species pre¬ sent in said test sample, said column bed compris¬ ing an equilibrated suspension of ion exchange ma- terial particles having a size less than 100 mesh, said particles in a column bed being one member selected from the class consisting of CarXH and CarYOH, where "Car" represents an inert substrate for carrying ionizable groups X providing disso- ciated cations H and ionizable groups Y provid¬ ing dissociated anions OH~, said CarXH particles being a weakly acidic cation exchanger having a reported pK : 3-7 and being used in an equilibra- ted suspension at a pH: 6.0-7.5 at 22.5°C, said CarYOH particles being a weakly basic anion ex¬ changer having a reported pK : 7-10 and being used in an equilibrated suspension at a pH: 7.3-9.0 at 22.5°C, aiid thereafter, a quantity of a buffer solution having no cyanide therein is introduced into an end of said column bed to preferentially elute therefrom a first fraction which contains certain of the hemoglobin species present in said test sample, a column bed which is an equilibrated suspension of CarXH particles providing a said first frac¬ tion containing substantially all of the hemo- globin species Hb-A, present in said test sam¬ ple, a column bed which is anequilibrated suspension of CarYOH particles providing a said first fraction containing essentially all of the hemoglobin spe- cies present in said test sample other than Hb-
A,la-c, and thereafter, an aliquot quantity of said first eluate fraction is collected from the other end of said column bed, and then, A quantity of a wash solution is intro¬ duced into an end of said column bed to desorb and elute therefrom a second fraction containing sub¬ stantially all of the remaining hemoglobin species present in said test sample, a column bed which is an equilibrated suspension of CarXH particles providing a said second fraction containing essen¬ tially all of the hemoglobin species present in said test sample other than Hb-A, J_&~c, a column bed which is an equilibrated suspension of CarYOH parti- cles providing a second fraction containing sub¬ stantially all of the hemoglobin species Hb-A, present in said test sample, and then, an aliquot quantity of said second elu¬ ate fraction is collected from -the other end of said column bed, and then, the hemoglobin species present in the said first and second eluate fractions are separate¬ ly detected and measured by spectrometric analysis and the respective amounts thereof are expressed as numerical values which are then compared in accordance with a mathematical formula to provide a numerical percentage value for the hemoglobin species Hb-A, in said test sample for use as a diagnostic indicator of the blood sugar charac¬ teristics of said specific person.
2. A method to determine a numerical percentage value as a diagnostic indicator of the blood sugar condition of a specific person, where¬ in a whole blood sample is taken from said person and thereafter prepared as a test sample contain¬ ing a red blood cell hemolysate solution, and thereafter, a quantity of said test sample is intro¬ duced into an end of a column bed having no cyanide therein which will adsorb hemoglobin species pre¬ sent in said test sample, said column bed compris¬ ing an equilibrated suspension of ion exchange material particles having a size less than 100 mesh, said particles in a column bed being one member selected from the class consisting of CarXH and
CarYOH, where "Car" represents an inert substrate for carrying ionizable groups X™* providing disso- ciated cations H + and ionizable groups Y+ providing dissociated anions 0H~, said CarXH particles being a weakly acidic cation exchanger having a reported pK : 3-7 and being used in an equilibrated suspen- sion at a pH: 6.0-7.5 at 22.5°C, said CarYOH parti¬ cles being a weakly basic anion exchanger having a reported pK 3.: 7-10 and being used in an equili- brated suspension at a pH: 7.3-9.0 at 22.5°C, and thereafter, a quantity of a buffer solution having no cyanide therein is introduced into an end of said column bed to preferentially elute therefrom a first fraction which contains certain of the hemo¬ globin species present in said test sample, a column bed which is an equilibrated suspension of CarXH particles providing a said first fraction containing substantially all of the hemoglobin species Hb-A, _ present in said test sample, a column bed which is an equilibrated suspension of CarYOH particles providing a said first fraction containing essentially all of the hemoglobin spe¬ cies present in said test sample other than ^-•A-ιa..c and thereafter, an aliquot quantity of said first eluate fraction is collected from the other end of said column bed, and then, a quantity of said test solution is sig- nificantly diluted to provide a red blood cell hemolysate solution which may be conveniently de¬ tected by spectrometric analysis, and then, the hemoglobin species present in said first eluate fraction and in the said diluted * hemolysate solution are separately detected and measured by spectrometric analysis and the respec¬ tive amounts thereof are expressed as numerical values which are then compared in accordance with a mathematical formula to provide a numerical per- centage value for the hemoglobin species Hb-A, in said test sample for use as a diagnostic indica¬ tor of the blood sugar characteristics of said spe¬ cific person.
3. A column bed for use in the method of either claim 1 or 2 comprising a suspension of CarYOH particles at an equilibrated pH of substantially 8.5 at 22.5°C and wherein "Car" is cellulosic and
Y is a diethylaminoethyl group
4. A column bed for use in the method of either claim 1 or 2 comprising a suspension of CarYOH particles at an equilibrated pH of sub¬ stantially 7.5 at 22.5°C and wherein "Car" is a resin copolymer of polystyrene and divinylbenzene and Y is a mixture of primary, secondary, and ter¬ tiary amine groups having the general formulae -NK2 , NHR, and N(R)„, where R is an aliphatic hydro¬ carbon radical such as -CH~ or -C-H,..
5. A column bed for use in the method of either claim 1 or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of sub¬ stantially 6.98 at 22.5°C and wherein "Car" is a resin copolymer of methacrylic acid and divinyl¬ benzene and X is a carboxyl group.
6. A column bed for use in the method of either claim 1 or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of sub¬ stantially 6.8 at 22.5°C and wherein "Car" is cellu- losic and X is a carboxymethyl group.
7. A column bed for use in the method of either claim 1 or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of substan¬ tially 6.1 at 22.5°C and wherein "Car" is cellu- losic and X is a carboxymethyl group.
8. A column bed for use in the method of either claim 1 or claim 2 comprising a suspension of CarXH particles at an equilibrated pH of sub¬ stantially 6.8 at 22.5°C and wherein "Car" is a resin copolymer of methacrylic acid and divinyl- benzene and X is a carboxyl group.
9. A microcolumn having no cyanide therein for use in the determination of a numerical percentage value as a diagnostic indicator of the blood sugar condition of a person providing a whole blood sample: having a reservoir discharging into a barrel terminating in a discharge tip, the junc¬ ture between said reservoir and said barrel and the juncture between said barrel and said tip each 5 being closed by a transverse disc, said discs being permeable to a red blood ceil hemolysate solution test sample prepared from said whole blood sample, and a column bed comprising an equilibrated sus¬ pension of ion exchange material particles having a 10 size less than 100 mesh positioned in said barrel between said discs, said particles in a column bed being one member selected from the class consisting of CarXH and CarYOH, where "Car" represents an inert substrate for carrying ionizable groups X providing
-f + 5 dissociated cations H and ionixable groups Y pro¬ viding dissociated anions OH , said CarXH particles being a weakly acidic cation exchanger having a reported pK : 3-7 and being used in an equilibrated suspension at a pH: 6.0-7.5 at 22.5°C, said CarYOH 0 particles being a weakly basic anion exchanger having a reported pK : 7-10 and being used in an equilibrated suspension at a pH: 7.3-9.0 at 22.5°C.
10. A column bed for the microcolumn of 5 claim 9 comprising a suspension of CarYOH particles at an equilibrated pH of substantially 8.5 at 2 222..55°°CC aanndd wwhheerreeiinn ""CCaarr"" is cellulosic and Y .+ is a diethy1aminoethy1 group.
0 11. A column bed for the microcolumn of claim 9 comprising a suspension of CarYOH parti¬ cles at an equilibrated pH of substantially 7.5 at 22.5°C and wherein "Car" is a resin copolymer of polystyrene and divinylbenzene and Y is a mixture -5 of primary, secondary and tertiary amine groups
Q having the general formulae -NIL*,, HR, and N(R)2, where R is an aliphatic hydrocarbon radical such as -CH3 or -C2H5.
12. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.98 at 22.5°C and wherein "Car" is a resin copolymer of methacrylic acid and divinylbenzene and X is a carboxyl group.
13. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.8 at 22.5'°C and wherein "Car" is cellulosic and X~ is a carboxymethyl group.
14. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.1 at
22.5°C and wherein "Car" is cellulosic and X is a carboxymethyl group.
15. A column bed for the microcolumn of claim 9 comprising a suspension of CarXH particles at an equilibrated pH of substantially 6.8 at 22.5°C and wherein "Car" is a resin copolymer of metha¬ crylic acid and divinylbenzene and X is a carboxyl group.
PCT/US1980/001411 1979-11-01 1980-10-22 Methods to determine a diagnostic indicator of blood sugar conditions,and,liquid chromatographic columns therefor(cyanide free) WO1981001245A1 (en)

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JP81500132A JPS6236181B2 (en) 1979-11-01 1981-12-02

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US06/090,319 US4238196A (en) 1979-11-01 1979-11-01 Methods to determine a diagnostic indicator of blood sugar conditions, and, liquid chromatographic columns therefor (cyanide free)
US90319 1987-08-27

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0392404A2 (en) * 1989-04-10 1990-10-17 Nippon Kayaku Kabushiki Kaisha Method for quantitatively measuring sugar-alcohol, column and kit for same
US5468380A (en) * 1989-04-26 1995-11-21 Nippon Kayaku Kabushiki Kaisha Method for quantitatively measuring sugar-alcohol, column and kit therefor

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1168705A (en) * 1980-05-27 1984-06-05 Hamish Small Ion exchange chromatography with indirect photometric detection
US4649122A (en) * 1980-09-02 1987-03-10 Leeco Diagnostics, Inc. Means for determining percentage of glycohemoglobin in whole blood
US4407961A (en) * 1981-03-16 1983-10-04 Sanders James L Ion-exchange system and method for isolation and determination of glycosylated hemoglobin in human blood
JPS58760A (en) * 1981-06-25 1983-01-05 Sekisui Chem Co Ltd Separation of catabolic hemoglobin
US4468330A (en) * 1981-04-27 1984-08-28 Sekisui Kagaku Kogyo Kabushiki Kaisha Filler for liquid chromatography useful for separating a hemoglobin variant in blood
WO1983003476A1 (en) * 1982-03-25 1983-10-13 James Lynn Sanders Ion-exchange system and method for isolation and determination of glycosylated hemoglobin in human blood
US4409335A (en) * 1982-05-28 1983-10-11 Bio-Rad Laboratories, Inc. Method for eliminating glucose dependent Schiff base effect from hemoglobin A1 assay
US4448888A (en) * 1982-08-24 1984-05-15 Bio-Rad Laboratories, Inc. Compositions and method for correcting temperature dependence of hemoglobin assays
US4935346A (en) 1986-08-13 1990-06-19 Lifescan, Inc. Minimum procedure system for the determination of analytes
US4787971A (en) * 1987-01-23 1988-11-29 Alan Donald Miniaturized column chromatography separation apparatus and method of assaying biomolecules employing the same
US4853338A (en) * 1987-05-20 1989-08-01 Technicon Instruments Corporation Cyanide-free hemoglobin reagent
GB2220369B (en) * 1988-06-10 1993-01-27 Inst Of Child Health Method for testing body fluids by low pressure liquid chromatography
US5807747A (en) * 1989-06-13 1998-09-15 Clinical Innovations Limited Method and apparatus for determination of glycosylated protein
JPH0486554A (en) * 1990-07-30 1992-03-19 Tosoh Corp Analysis method for sugar albumin
EP1239287A3 (en) * 1992-03-04 2006-07-26 Abbott Laboratories Composition for use as an ion capture separation reactant
FI112545B (en) * 1997-05-30 2003-12-15 Nokia Corp Method and system for predicting the level of a glycosylated hemoglobin component in a patient's blood
US6177009B1 (en) * 1998-04-03 2001-01-23 Macherey, Nagel Gmbh & Co. Apparatus for treating biomolecules
US6458326B1 (en) 1999-11-24 2002-10-01 Home Diagnostics, Inc. Protective test strip platform
US6541266B2 (en) 2001-02-28 2003-04-01 Home Diagnostics, Inc. Method for determining concentration of an analyte in a test strip
US6525330B2 (en) 2001-02-28 2003-02-25 Home Diagnostics, Inc. Method of strip insertion detection
US6562625B2 (en) 2001-02-28 2003-05-13 Home Diagnostics, Inc. Distinguishing test types through spectral analysis
US6761855B1 (en) * 2001-11-05 2004-07-13 Biosearch Technologies, Inc. Column for solid phase processing

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4142857A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor
US4142858A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor
US4142856A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor
US4142855A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5499496A (en) * 1977-12-02 1979-08-06 Isolab Inc Method of deciding diagnosis index of blood sugar condition and its device

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4142857A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor
US4142858A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor
US4142856A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor
US4142855A (en) * 1977-12-02 1979-03-06 Isolab, Incorporated Method to determine a diagnostic indicator of blood sugar condition, and, a liquid chromatographic microcolumn therefor

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0392404A2 (en) * 1989-04-10 1990-10-17 Nippon Kayaku Kabushiki Kaisha Method for quantitatively measuring sugar-alcohol, column and kit for same
EP0392404A3 (en) * 1989-04-10 1992-07-08 Nippon Kayaku Kabushiki Kaisha Method for quantitatively measuring sugar-alcohol, column and kit for same
US5468380A (en) * 1989-04-26 1995-11-21 Nippon Kayaku Kabushiki Kaisha Method for quantitatively measuring sugar-alcohol, column and kit therefor

Also Published As

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GB2074466A (en) 1981-11-04
GB2074466B (en) 1983-08-03
JPS57501937A (en) 1982-10-28
JPS6236181B2 (en) 1987-08-05
US4238196A (en) 1980-12-09
CA1110467A (en) 1981-10-13
CH659527A5 (en) 1987-01-30

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