Methods, Devices, and Systems for Glycated Hemoglobin Analysis
Methods, devices, and systems for measuring the concentration of glycated hemoglobin (HbA 1 c) in a blood sample are provided. A blood sample may be contacted with a lysing agent, an assay to measure total Hb, and an assay to measure HbA 1 c, which comprises donor and acceptor dyes attached to a Hb specific antibody and a HbA 1 c analog. Preferably, the emission spectrum of the donor dye overlaps with the excitation spectrum of the acceptor dye. The concentration of HbA 1 c in the sample may be correlated with an increase in donor dye and/or a decrease in acceptor dye fluorescence in the presence of HbA 1 c as compared to the donor and/or the acceptor dye fluorescence in the absence of HbA 1 c.
1 . A method for determining the concentration of glycated hemoglobin (HbA1c) in a sample, comprising:
contacting the sample with a red blood cell lysing mixture;
contacting the sample with a total hemoglobin assay;
contacting the sample with a HbA1c assay including one of
a fluorescent donor dye conjugated to a HbA1c analog and a fluorescent acceptor dye attached to a Hb-specific antibody, and
a fluorescent donor dye attached to a Hb-specific antibody and a fluorescent acceptor dye attached to a HbA1c analog,
where the emission spectrum of the fluorescent donor dye overlaps with the excitation spectrum of the fluorescent acceptor dye; and
correlating the concentration of HbA1c in the sample with a change in the intensity of at least one of donor dye fluorescence and acceptor dye fluorescence.
2 . The method of claim 1 , where the total hemoglobin assay comprises:
converting hemoglobin to a metal-hemoglobin complex; and
detecting the intensity of light responsive to the metal-hemoglobin complex.
3 . The method of claim 1 , the fluorescent donor dye having an absorption maximum at an excitation wavelength from about 590 to about 660 nanometers.
4 . The method of claim 1 , the fluorescent acceptor dye having an absorption maximum at an excitation wavelength from about 630 to about 760 nanometers.
5 . The method of claim 1 , where the fluorescent donor dye is fluorescein isothiocyanate and the fluorescent acceptor dye is tetramethylrhodamine isothiocyanate.
6 . The method of claim 1 , where the HbA1c analog is any molecule that binds to the HbA1c-specific antibody with less affinity than HbA1c.
7 . The method of claim 1 , where the HbA1c analog is selected from the group consisting of glycated peptides, multiple antigen peptides, and combinations thereof.
8 . The method of claim 1 , where the fluorescence of the fluorescent donor dye and the fluorescence of the fluorescent acceptor dye are detected at a wavelength from about 450 to about 520 nanometers or greater than about 600 nanometers.
9 . The method of claim 1 , the fluorescent acceptor dye replaced with a quenching dye.
10 . The method of claim 1 , where the change in the intensity includes an increase in the intensity of the donor dye fluorescence and a decrease in the intensity of the acceptor dye fluorescence.
11 . The method of claim 1 , where the change in the intensity includes a decrease in the intensity of the donor dye fluorescence and an increase in the intensity of the acceptor dye fluorescence.
12 . A hemoglobin ratio determination system, comprising:
a vial comprising a lysing mixture;
a sensor strip,
where a total hemoglobin assay is included with the lysing mixture or the sensor strip,
where a HbA1c assay is included with the lysing mixture or the sensor strip, and where the HbA1c assay includes
a fluorescent donor dye attached to a HbA1c analog and a fluorescent acceptor dye attached to an Hb-specific antibody, or
a fluorescent donor dye attached to a Hb-specific antibody and a fluorescent acceptor dye attached to a HbA1c analog, and where
an emission spectrum of the fluorescent donor dye overlaps with an excitation spectrum of the fluorescent acceptor dye; and
an electronic measurement device including a processor, where the processor is operable to determine a ratio of HbA1c to total hemoglobin.
13 . The system of claim 12 , where the processor determines a comparison of the decrease in the acceptor dye fluorescence in the presence of HbA1c to the acceptor dye fluorescence in the absence of HbA1c.
14 . The system of claim 12 , where the processor determines a comparison of the increase in donor dye fluorescence in the presence of HbA1c to the donor dye fluorescence in the absence of HbA1c.
15 . The system of claim 13 , where the processor determines a correlation of HbA1c in the sample responsive to the comparison and the ratio of HbA1c to total hemoglobin is responsive to the correlation.
16 . The system of claim 15 , where the processor determines the total hemoglobin in the sample substantially before determining the HbA1c in the sample.
17 . The system of claim 12 , the fluorescent acceptor dye replaced with a quenching dye.
18 . A HbA1c sample concentration determination kit, comprising:
a vial including a lysing mixture, a total Hb assay, and a HbA1c assay, the HbA1c assay including one of
a fluorescent donor dye attached to a HbA1c analog and a fluorescent acceptor dye attached to an Hb-specific antibody, and
a fluorescent donor dye attached to a Hb-specific antibody and a fluorescent acceptor dye attached to a HbA1c analog; and where
an emission spectrum of the donor dye overlaps with an excitation spectrum of the acceptor dye.
19 . The kit of claim 18 , the fluorescent acceptor dye replaced with a quenching dye.
20 . A HbA1c sample concentration determination kit, comprising:
a vial including a lysing mixture and a total Hb assay, and
a sensor strip including a HbA1c assay, the HbA1c assay including one of
a fluorescent donor dye attached to a HbA1c analog and a fluorescent acceptor dye attached to an Hb-specific antibody, and
a fluorescent donor dye attached to a Hb-specific antibody and a fluorescent acceptor dye attached to a HbA1c analog; and where
an emission spectrum of the donor dye overlaps with an excitation spectrum of the acceptor dye.
21 . The kit of claim 20 , the fluorescent acceptor dye replaced with a quenching dye.