Reference method variability. A bias plot was generated to determine the variability of the reference method when patient samples were measured in duplicate. (A) Visual inspection of the data showed that both measurements closely follow the equality line. (B) On the x-axis of the bias plot is the mean value of the duplicates and on the y-axis is the difference between the 2 measurements. The dotted line is the estimated mean bias from 0 between the measurements. If there was no difference in the mean bias this line would be at 0, which it is. The dashed lines are the 95% limits of agreement, which indicates that 95% of the second measurements were within approximately plus or minus 2.5-fold of the first measurement. Ninety percent of the values were within 2-fold.