Abstract
Determination method of the HbA1 by electrophoresis (1) using the Agar-Gel Film developed by Coming was investigated.
1) Reagents and Apparatuses Buffer Solution: 0.1M Citrate buffer, pH 6.2 Support Medium: HbA1 Agar-Gel Film (Corning) Hemolyzing Agent: 0.1% Saponin and 0.05% EDTA Electrophoresis Chamber: U-shaped electrophoresis chamber Densitometer: M-720 Densitometer (Corning)
2) Measuring Method The hemolysate is obtained by adding 300 μl of saponin to 100μl of blood collected in EDTA. 1μl of hemolysate thus prepared is applied to the Agar-Gel film and is electrified at 60V for 40 minutes. The film is then dessicated in the oven and is put on the densitometer at 420nm for obtaining the HbA1 value.
3) Results
The within-day precision of this method was x=7.26%, C. V. =2.20% in healthy subjects and =10.71%, C. V.=1.69% in diabetic patients.(Table 1) The between-day precision was x=7.09% x, C. V. =2.76% in healthy subjects and x=9.44%, C. V. 1.79% in diabetic patients.(Fig. 1)
The blood sample collected in EDTA was immediately hemolyzed. The hemolysate thus prepared was kept at 4°. No fluctuation was observed on HbA1 value for 14 days.
No significant difference was noted among the HbA1 value at 4°, 10°, 20° and 37° of buffer solution.(Fig. 2)
It is known that the disposable columns (mini-column) allow bilirubin to influence on the measurement value, whereas no influence was noted up to its amount of 30mg/dl in this method.
Comparative studies were made on the blood sample for HbA1 measurement. The average values in healthy subjects were x=6.76% with disposable columns (2)(mini-column) and Y=6.93% with this method. The correlation coefficient between the two methods was γ=0.96 with the regression equation, Y=0.90X+0.89. The average value in diabetic patients were x=9.75% with disposable columns and Y=10.05% with this method. The correlation coefficient between the two methods was γ=0.98 with the regression eqaution, Y=1.10X-0.68.(Fig. 3)
The average values in healthy subjects were x=6.69% with the column method (3) and Y=6.94% with this method. The correlation coefficient between the two methods was γ=0.96 with the regression equation, Y=0.98X+0.41. The average value in diabetic patient was x=9.60% with column method and Y=10.05% with this method. The correlation coefficient between the two methods was γ=0.97 with the regression equation, Y=1.12X+0.66.(Fig. 4)
The average HbA1 value was x=4.70% with the column method (3) and the average HbA1 value was Y=6.94% with this method both in healthy subject. The correlation coefficient between the two months was γ=0.96 with the regression equation, Y=1.03X+2.11. The average HbA1C value was x=7.49% with the column method and the average HbA1 value as Y=10.05% with this method in diabetic patient. The correlation coefficient between the two methods was γ=0.96 with the regression equation, Y=1.12X+1.66.(Fig. 5)
4) Conclusion
From the aforementioned experimental results, it can be said that the Agar-Gel Electrophoresis, which requires so less amount of sample as 100μl and gives the favorable reproductibility, is simple, time-saving, satisfactorily endurable to the influences of the temperature and bilirubin, and highly correlated with the disposable column method or column method. Therefore, it can conclusively be assessed that this method is an attractive and useful alternative to the other methods for the routine measurement of HbA1.
