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

• US 4,238,196 A
• Filed: 11/01/1979
• Issued: 12/09/1980
• Est. Priority Date: 11/01/1979
• Status: Expired due to Term

• Current Assignee: Wallac, Inc. (Revvity, Inc.)
• Original Assignee: Isolab Inc. (Revvity, Inc.)
• Inventors: Rosenthal, Murray A., Acuff, Kenneth J., Volk, Murray E.
• Primary Examiner(s): Adee, John
• Application Number:
  US06/090,319
• Time in Patent Office: 404 Days
• Field of Search: 210/31 C, 210/198 C, 210/DIG. 23, 23/230 B, 422/70
• US Class Current: 436/67
• CPC Class Codes: B01D 15/08 Selective adsorption, e.g. chromatography
• CPC Class Codes: B01D 15/362 Cation-exchange
• CPC Class Codes: B01D 15/363 Anion-exchange
• CPC Class Codes: G01N 30/02 Column chromatography
• CPC Class Codes: G01N 30/6004 end pieces
• CPC Class Codes: G01N 30/96 using ion-exchange G01N30/02, G01N30/90 take precedence
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: H01M 50/182 for cells with a collector centrally disposed in the active mass, e.g. Leclanché cells
• CPC Class Codes: H01M 50/342 Non-re-sealable arrangements
• CPC Class Codes: H01M 50/375 Vent means sensitive to or responsive to temperature
• CPC Class Codes: Y02E 60/10 Energy storage using batteries

Abstract

Methods and microcolumns to determine a numerical percentage value as a diagnostic indicator of the blood sugar condition of a specific person. A whole blood sample is lysed and a test sample thereof is placed on a microchromatographic column bed of ion exchange material particles. 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 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. A mathematical computation using integer factors corresponding to amounts of various hemoglobin species produces the numerical percentage value.

Claims

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 thereafter,

a quantity of said test sample is introduced into an end of a column bed having no cyanide therein which will adsorb hemoglobin species present in said test sample, said column bed comprising 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 X³¹ providing dissociated cations H⁺ and ionizable groups Y⁺ providing dissociated anions OH^-, said CarXH particles being a weakly acidic cation exchanger having a reported pK_a :

3-7 and being used in an equilibrated suspension at a pH;

6.0-7.5 at 22.5°

C., said CarYOH particles being a weakly basic anion exchanger having a reported pK_a ;

7-10 and being used in an equilibrated 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 hemoglobin 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_1a-c 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 species present in said test sample other than Hb-A_1a-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 introduced into an end of said column bed to desorb and elute therefrom a second fraction containing substantially all of the reminaing hemoglobin species present in said test sample, a column bed which is an equilibrated suspension of CarXH particles providing a said second fraction containing essentially all of the hemoglobin species present in said test sample other than Hb-A_1a-c, a column bed which is an equilibrated suspension of CarYOH particles providing a said second fraction containing substantially all of the hemoglobin species Hb-A_1a-c present in said test sample, and then,

an aliquot quantity of said second eluate 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 separately 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_1a-c in said test sample for use as a diagnostic indicator of the blood sugar characteristics of said specific 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 substantially 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 tertiary amine groups having the general formulae --NH₂, NHR, and N(R)₂, where R is an aliphatic hydrocarbon radical such as --CH₃ or --C₂ H₅.

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 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.

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 substantially 6.8 at 22.5°

C. and wherein "Car" is cellulosic 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 substantially 6.1 at 22.5°

C. and wherein "Car" is cellulosic 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 substantially 6.8 at 22.5°

C. and wherein "Car" is a resin copolymer of methacrylic acid and divinylbenzene and X^- is a carboxyl group.

2. 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 thereafter,

a quantity of said test sample is introduced into an end of a column bed having no cyanide therein which will adsorb hemoglobin species present in said test sample, said column bed comprising 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 dissociated cations H⁺ and ionizable groups Y⁺ providing dissociated anions OH^-, said CarXH particles being a weakly acidic cation exchanger having a reported pK_a :

3-7 and being used in an equilibrated suspension at a pH;

6.0-7.5 at 22.5°

C., said CarYOH particles being a weakly basic anion exchanger having a reported pK_a ;

7-10 and being used in an equilibrated 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 hemoglobin 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_1a-c 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 species present in said test sample other than Hb-A_1a-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 significantly diluted to provide a red blood cell hemolysate solution which may be conveniently detected 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 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_1a-c in said test sample for use as a diagnostic indicator of the blood sugar characteristics of said specific person.

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 juncture between said reservoir and said barrel and the juncture between said barrel and said tip each being closed by a transverse disc, said discs being permeable to a red blood cell hemolysate solution test sample prepared from said whole blood sample, and a column bed comprising an equilibrated suspension of ion exchange material particles having a 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 dissociated cations H⁺ and ionizable groups Y⁺ providing dissociated anions OH^-, said CarXH particles being a weakly acidic cation exchanger having a reported pK_a ;

3-7 and being used in an equilibrated suspension at a pH;

6.0-7.5 at 22.5°

C., said CarYOH particles being a weakly basic anion exchanger having a reported pK_a ;

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 claim 9 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.

11. A column bed for the microcolumn of claim 9 comprising a suspension of CarYOH particles 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 of primary, secondary and tertiary amine groups having the general formulae --NH₂, NHR, and N(R)₂, where R is an aliphatic hydrocarbon radical such as --CH₃ or --C₂ H₅.

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 methacrylic acid and divinylbenzene and X^- is a carboxyl group.