Question

1-4

Pipette 0.1 ml serum and 0.3 mL DI water to a small tube and mix. I assay this diluted serum and obtain a glucose value of 860 mg/ Liter. The glucose value I will report is ??? How do you know when an assay must be repeated on dilution? How do you find the value for the limit of linearity for a method? I read the absorbance for the patient's sample on instrument #2. I used a calibration curve made using spectrophotometer #4 to read the glucose values. Is this acceptable? Discuss.

Answer 1

1) Diluting 0.1 mL serum in 0.3 mL DI water is equivalent to a 1 in 4 dilution (1:4). The dilution factor is therefore 4. We are told that when this diluted mix was assayed for serum glucose, we obtained a value of 860 mg/L. The glucose value that has to be reported has to be corrected for the reduction caused by the dilution. This is done by multiplying the readout with the dilution factor. Here the dilution factor is 4, so the value that must be reported is 4 x 860 = 3440 mg/L.

2) Most assays have a linear range within the concentration of the substance being assayed exhibits a linear relationship with the absorbance or transmittance measured by the spectrophotometer (this relationship can be fitted into the form of y = mx+c, where x is the concentration and y is the absorbance/transmittance). A good estimation of the sample concentration (of the substance being assayed) can be made only within this linear range. If it is suspected that the concentration of the substance to be assayed lies beyond this range, the sample has to be diluted to bring the concentration within this linear range.

3) The limit of linearity can be determined by creating a calibration curve for the substance to be assayed in a medium that is the same or almost similar to the sample that has to be assayed. A sample matrix is the components of the sample other than the analyte of interest. In our case, if we are measuring serum glucose levels, our analyte of interest is glucose, and the matrix is constituted of all other salts and protein components of the serum. Know concentrations of glucose can be dissolved in glucose free serum or serum substitute to make the calibration curve.

4) This would not be acceptable as instruments may vary in their sensitivity and other parameters that can affect the estimation. The most accurate estimations can be obtained only if the calibration curve and the sample measurements are recorded using the same instrument with the same kind of matrix and cuvettes.

I hope this helps :)