Method for determination of glucose concentration in whole blood
First Claim
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1. A method for the in vitro measurement of the concentration of glucose in a whole blood sample, which comprises:
- A) obtaining a first whole blood sample and separating it into its serum and cellular fractions;
B) mixing samples of the separated serum and cellular fractions of step (A) in varying proportions;
C) diluting each of the samples obtained from step (B) in a vessel with a solution suitable for measuring the glucose concentration in the diluted sample;
D) obtaining a first value (Cy) of the concentration of glucose in each of said diluted samples before the glucose contained within the cells has diffused out of the cells into the solution used to dilute the sample;
E) obtaining a second value (Cx) of the concentration of glucose in each of said diluted samples after the glucose contained within the cells has diffused out of the cells into the solution used to dilute the sample;
F) creating a calibration curve relating the proportion of the whole blood sample which comprises the serum fraction (X) to the ratio of the value of the step (E) to the value of step (D) (Cx/Cy);
G) creating a calibration curve relating a conversion factor (Co/Cx), said conversion factor being the ratio of Cx when X is 100% (Co) to the Cx obtained at variable values of X, to the proportion of the whole blood volume which consists of the volume of the cellular fraction (1-X);
H) obtaining a direct relationship between the conversion factor (Co/Cx) and (Cx/Cy) by mathematically eliminating the serum fraction (X) from the calibration curves obtained in steps (F) and (G);
I) obtaining a second whole blood sample in which the glucose concentration is to be determined;
J) diluting said second whole blood sample in a solution suitable for the measurement of glucose concentration in said diluted second whole blood sample;
K) obtaining a first value of the concentration of glucose in the sample solution obtained in step (J) before the glucose contained within the cells has diffused out of the cells into the solution used to dilute the second whole blood sample;
L) obtaining a second value of the concentration of glucose in the sample solution obtained in step (J) after the glucose contained within the cells has diffused out of the cells into the solution used to dilute the second whole blood sample;
M) obtaining the ratio of Cx/Cy from the values obtained in steps (K) and (L);
N) obtaining the value for the conversion factor (Co/Cx) from the relationship obtained in step (H) with reference to the ratio Cx/Cy obtained in step (M); and
O) determining the concentration of glucose in the whole blood sample of step (I) by multiplying the value of Cx obtained in step (L) by the value of the conversion factor obtained in step (N).
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Abstract
There is provided a method for the determination of a glucose concentration in a whole blood utilizing a biosensor. A correction of the measured glucose concentration for dilution error introduced by the solid component of blood cells is calculated based on the change in glucose concentration measured before and after significant glucose has diffused from blood cells into the buffer used to dilute the sample. Thus, the need to centrifuge blood samples to obtain a cell-free serum sample for glucose determination is eliminated.
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19 Claims
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1. A method for the in vitro measurement of the concentration of glucose in a whole blood sample, which comprises:
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A) obtaining a first whole blood sample and separating it into its serum and cellular fractions; B) mixing samples of the separated serum and cellular fractions of step (A) in varying proportions; C) diluting each of the samples obtained from step (B) in a vessel with a solution suitable for measuring the glucose concentration in the diluted sample; D) obtaining a first value (Cy) of the concentration of glucose in each of said diluted samples before the glucose contained within the cells has diffused out of the cells into the solution used to dilute the sample; E) obtaining a second value (Cx) of the concentration of glucose in each of said diluted samples after the glucose contained within the cells has diffused out of the cells into the solution used to dilute the sample; F) creating a calibration curve relating the proportion of the whole blood sample which comprises the serum fraction (X) to the ratio of the value of the step (E) to the value of step (D) (Cx/Cy); G) creating a calibration curve relating a conversion factor (Co/Cx), said conversion factor being the ratio of Cx when X is 100% (Co) to the Cx obtained at variable values of X, to the proportion of the whole blood volume which consists of the volume of the cellular fraction (1-X); H) obtaining a direct relationship between the conversion factor (Co/Cx) and (Cx/Cy) by mathematically eliminating the serum fraction (X) from the calibration curves obtained in steps (F) and (G); I) obtaining a second whole blood sample in which the glucose concentration is to be determined; J) diluting said second whole blood sample in a solution suitable for the measurement of glucose concentration in said diluted second whole blood sample; K) obtaining a first value of the concentration of glucose in the sample solution obtained in step (J) before the glucose contained within the cells has diffused out of the cells into the solution used to dilute the second whole blood sample; L) obtaining a second value of the concentration of glucose in the sample solution obtained in step (J) after the glucose contained within the cells has diffused out of the cells into the solution used to dilute the second whole blood sample; M) obtaining the ratio of Cx/Cy from the values obtained in steps (K) and (L); N) obtaining the value for the conversion factor (Co/Cx) from the relationship obtained in step (H) with reference to the ratio Cx/Cy obtained in step (M); and O) determining the concentration of glucose in the whole blood sample of step (I) by multiplying the value of Cx obtained in step (L) by the value of the conversion factor obtained in step (N). - View Dependent Claims (2, 3, 4, 5)
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6. A method for the measurement of the concentration of glucose in a whole blood sample, by means of a sensor comprising a hydrogen peroxide electrode (HPE), the output of which is a function of the progress of a glucose oxidase enzymatic reaction occurring in a vessel, which comprises:
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A) diluting a standard solution of defined glucose concentration in a buffer in a reaction vessel containing glucose oxidase and a HPE, and obtaining a calibration curve relating the current of the HPE, measured when the rate of hydrogen peroxide production has reached a steady state, to the concentration of glucose; B) diluting a standard solution of defined glucose concentration with said buffer in said reaction vessel, and obtaining a calibration curve relating the maximum value of the first derivative with respect to time of the current of the HPE to the concentration of glucose; C) obtaining a first whole blood sample and separating it into the serum and cellular fractions; D) mixing samples of the separated fractions of step (C) in varying proportions; E) diluting each of the samples obtained from step (D) in a buffer suitable for measuring the glucose concentration in the diluted sample; F) obtaining a first reading in each of the diluted samples of step (E), of the current generated by the HPE after the rate of hydrogen peroxide production has reached a steady state, then converting said first reading into a first measurement of the glucose concentration in said diluted samples of step (E) by reference to the calibration curve obtained in step (A), the value of said measurement being called Cx; G) obtaining a second reading in each of the diluted samples of step (E), of the maximum value of the first derivative with respect to time of the current generated by the HPE, then converting said second reading into a second measurement of the glucose concentration in said diluted samples of step (E) by reference to the calibration curve obtained in step (B), the value of said measurement being called Cy; H) creating a calibration curve relating the proportion of the whole blood sample which comprises the serum fraction (X) to the ratio of the measurement of step (F) to the measurement of step (G) (Cx/Cy); I) creating a calibration curve relating a conversion factor (Co/Cx), said conversion factor being the ratio of Cx when X is 100% (Co) to the Cx obtained at variable values of X (Co/Cx) to the proportion of the whole blood volume which consists of the volume of the cellular fraction (1-X); J) obtaining a direct relationship between the conversion factor (Co/Cx) and (Cx/Cy) by mathematically eliminating the serum fraction (X) from the calibration curves obtained in steps (H) and (I); K) obtaining a second whole blood sample, in which the glucose concentration is to be determined, and diluting said second whole blood sample with said buffer in said reaction vessel; L) obtaining a first reading in said whole blood sample of step (K), of the current generated by the HPE after the rate of hydrogen peroxide production has reached a steady state, then converting said first reading into a first measurement of the glucose concentration in said second whole blood sample by reference to the calibration curve obtained in step (A), the value of said measurement being called Cx; M) obtaining a second reading in said second whole blood sample of step (K), of the maximum value of the first derivative with respect to time of the current generated by the HPE, then converting said second reading into a second measurement of the glucose concentration in said second whole blood sample by reference to the calibration curve obtained in step (B), the value of said measurement being called Cy; N) determining the conversion factor (Co/Cx) by reference to the relationship obtained in step (J) with reference to Cx and Cy obtained in steps (L) and (K), respectively; and o) multiplying said first measurement of step (L), Cx, by the conversion factor obtained in step (N) (Co/Cx) to obtain the value of the true glucose concentration in the whole blood of step (K). - View Dependent Claims (7, 8)
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9. A method for the in vitro measurement of the concentration of glucose in a whole blood sample, which comprises:
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A) obtaining a first whole blood sample and separating it into its serum and cellular fractions; B) mixing samples of the separated serum and cellular fractions of step (A) in varying proportions; C) diluting each of the samples obtained from step (B) in a vessel with a solution suitable for measuring the glucose concentration in the diluted sample; D) obtaining a first value (Cy) of the concentration of glucose in each of said diluted samples before the glucose contained within the cells has diffused out of the cells into the solution used to dilute the sample; E) obtaining a first value (Cx) of the concentration of glucose in each of said diluted samples after the glucose contained within the cells has diffused out of the cells into the solution used to dilute the sample; F) creating a calibration curve relating the proportion of the whole blood sample which comprises the serum fraction (X) to the ratio of the value of step (E) to the value of step (D) (Cx/Cy); G) creating a calibration curve relating a conversion factor (Co/Cy), said conversion factor being the ratio of Cy when X is 100% (Co) to the Cy obtained at variable values of X, to the proportion of the whole blood volume which consists of the volume of the cellular fraction (1-X); H) obtaining a direct relationship between the conversion factor (Co/Cy) and (Cx/Cy) by mathematically eliminating the serum fraction (X) from the calibration curves obtained in steps (F) and (G); I) obtaining a second whole blood sample in which the glucose concentration is to be determined; J) diluting said second whole blood sample in a solution suitable for the measurement of glucose concentration in said diluted second whole blood sample; K) obtaining a first value (Cy) of the concentration of glucose in the sample solution obtained in step (J) before the glucose contained within the cells has diffused out of the cells into the solution used to dilute the second whole blood sample; L) obtaining a second value (Cx) of the concentration of glucose in the sample solution obtained in step (J) after the glucose contained within the cells has diffused out of the cells into the solution used to dilute the second whole blood sample; M) obtaining the ratio of Cx/Cy from the values obtained in steps (K) and (L); N) obtaining the value for the conversion factor (Co/Cy) from the relationship obtained in step (H) with reference to the ratio Cx/Cy obtained in step (M); and O) determining the concentration of glucose in the whole blood sample of step (I) by multiplying the value of Cy obtained in step (K) by the value of the conversion factor obtained in step (N). - View Dependent Claims (10, 11, 12, 13, 14)
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15. A method for the measurement of the concentration of glucose in a whole blood sample, by means of a sensor, the output of which is a function of the progress of a glucose oxidase enzymatic reaction occurring in a vessel, which comprises:
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A) diluting a standard solution of defined glucose concentration with a buffer in a reaction vessel containing glucose oxidase and the sensor, and obtaining a calibration curve relating the output of the sensor, measured when the rate of hydrogen peroxide production has reached a steady state, to the concentration of glucose; B) diluting a standard solution of defined glucose concentration with said buffer in said reaction vessel, and obtaining a calibration curve relating the maximum value of the first derivative with respect to time of the output of the sensor to the concentration of glucose; C) obtaining a first whole blood sample and separating it into the serum and cellular fractions; D) mixing samples of the separated fractions of step (C) in varying proportions; E) diluting each of the samples obtained from step (D) in a buffer suitable for measuring the glucose concentration in the diluted sample; F) obtaining a first reading in each of the diluted samples of step (E), of the output of the sensor after the rate of hydrogen peroxide production has reached a steady state, then converting said first reading into a first measurement of the glucose concentration in said diluted samples of step (E) by reference to the calibration curve obtained in step (A), the value of said measurement being called Cx; G) obtaining a second reading in each of the diluted samples of step (E), of the maximum value of the first derivative with respect to time of the output of the sensor, then converting said second reading into a second measurement of the glucose concentration in said diluted samples of step (E) by reference to the calibration curve obtained in step (B), the value of said measurement being called Cy; H) creating a calibration curve relating the proportion of the whole blood sample which comprises the serum fraction (X) to the ratio of the measurement of step (F) to the measurement of step (G) (Cx/Cy); I) creating a calibration curve relating a conversion factor (Co/Cx), said conversion factor being the ratio of Cx when X is 100% (Co) to the Cx obtained at variable values of X (Co/Cx) to the proportion of the whole blood volume which consists of the volume of the cellular fraction (1-X); J) obtaining a direct relationship between the conversion factor (Co/Cx) and (Cx/Cy) by mathematically eliminating the serum fraction (X) from the calibration curves obtained in steps (H) and (I); K) obtaining a second whole blood sample, in which the glucose concentration is to be determined, and diluting said whole blood sample with said buffer in said reaction vessel; L) obtaining a first reading in said whole blood sample of step (K), of the output of the sensor after the rate of hydrogen peroxide production has reached a steady state, then converting said first reading into a first measurement of the glucose concentration in said second whole blood sample by reference to the calibration curve obtained in step (A), the value of said measurement being called Cx; M) obtaining a second reading in said second whole blood sample of step (K), of the maximum value of the first derivative with respect to time of the output of the sensor, then converting said second reading into a second measurement of the glucose concentration in said second whole blood sample by reference to the calibration curve obtained in step (B), the value of said measurement being called Cy; N) determining the conversion factor (Co/Cx) by reference to the relationship obtained in step (J) with reference to Cx and Cy obtained in steps (L) and (K), respectively; and O) multiplying said first measurement of step (L), Cx, by the conversion factor obtained in step (N) (Co/Cx) to obtain the value of the true glucose concentration in the whole blood of step (K). - View Dependent Claims (16, 17)
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18. A method for the measurement of the concentration of glucose in a whole blood sample, by means of a sensor, the output of which is a function of the progress of a glucose oxidase enzymatic reaction occurring in a vessel, which comprises:
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A) diluting a standard solution of defined glucose concentration with a buffer in a reaction vessel containing glucose oxidase and the sensor, and obtaining a calibration curve relating the output of the sensor, measured when the rate of hydrogen peroxide production has reached a steady state, to the concentration of glucose; B) diluting a standard solution of defined glucose concentration with said buffer in said reaction vessel, and obtaining a calibration curve relating the maximum value of the first derivative with respect to time of the output of the sensor to the concentration of glucose; C) obtaining a first whole blood sample and separating it into the serum and cellular fractions; D) mixing samples of the separated fractions of step (C) in varying proportions; E) diluting each of the samples obtained from step (D) in a buffer suitable for measuring the glucose concentration in the diluted sample; F) obtaining a first reading in each of the diluted samples of step (E), of the output of the sensor after the rate of hydrogen peroxide production has reached a steady state, then converting said first reading into a first measurement of the glucose concentration in said diluted samples of step (E) by reference to the calibration curve obtained in step (A), the value of said measurement being called Cx; G) obtaining a second reading in each of the diluted samples of step (E), of the maximum value of the first derivative with respect to time of the output of the sensor, then converting said second reading into a second measurement of the glucose concentration in said diluted samples of step (E) by reference to the calibration curve obtained in step (B), the value of said measurement being called Cy; H) creating a calibration curve relating the proportion of the whole blood sample which comprises the serum fraction (X) to the ratio of the measurement of step (F) to the measurement of step (G) (Cx/Cy); I) creating a calibration curve relating a conversion factor (Co/Cy), said conversion factor being the ratio of Cy when X is 100% (Co) to the Cy obtained at variable values of X (Co/Cy) to the proportion of the whole blood volume which consists of the volume of the cellular fraction (1-X); J) obtaining a direct relationship between the conversion factor (Co/Cy) and (Cx/Cy) by mathematically eliminating the serum fraction (X) from the calibration curves obtained in steps (H) and (I); K) obtaining a second whole blood sample, in which the glucose concentration is to be determined, and diluting said second whole blood sample with said buffer in said reaction vessel; L) obtaining a first reading in said whole blood sample of step (K), of the output of the sensor after the rate of hydrogen peroxide production has reached a steady state, then converting said first reading into a first measurement of the glucose concentration in said second whole blood sample by reference to the calibration curve obtained in step (A), the value of said measurement being called Cx; M) obtaining a second reading in said second whole blood sample of step (K), of the maximum value of the first derivative with respect to time of the output of the sensor, then converting said second reading into a second measurement of the glucose concentration in said second whole blood sample by reference to the calibration curve obtained in step (B), the value of said measurement being called Cy; N) determining the conversion factor (Co/Cy) by reference to the relationship obtained in step (J) with reference to Cx and Cy obtained in steps (L) and (K), respectively; and O) multiplying said second measurement of step (M), Cy, by the conversion factor obtained in step (N) (Co/Cy) to obtain the value of the true glucose concentration in the whole blood of step (K). - View Dependent Claims (19)
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Specification