We already know it’s important to know the difference between the “true” quantity of any given substance and the individual measurement result. What that means is that we need to know two distinct things: the limit of the blank (LoB) and the limit of detection (LoD). CLSI Protocols teach us that knowing these values is important — especially when it comes to knowing the difference between the both of them.
The lowest assay limit is considered the limit of the blank. This is also defined as the highest value we could likely expect to see from a set of measurements on a sample that specifically has no analyte. In other words, the true concentration of the analyte is zero. The limit of detection, on the other hand, is the analyte concentration at which a sample measurement or test result may actually exceed the limit of the blank and become detectable. Hence the term “limit of detection.”
Unfortunately, many people use a shortcut to determine the limit of detection in their test results. This practice involves determining the limit of detection by measuring a “blank” sample multiple times and then determining the limit of detection by adding the mean and 3SD together. While the shortcut may seem efficient, CLSI documentation notes that this method actually doesn’t allow anyone to differentiate a true analyte quantity.
Fortunately for us, CLSI documentation helps distinguish between multiple “limits” to help provide clarity in the measurement process. The lower limit of the linear range encompasses the lowest measured value of concentration that maintains a linear relationship with the true concentration, or value. We can determine this value via a linearity experiment. The low end of the analytical measurement range, however, is the lowest concentration that meets our defined parameters. These parameters can include things like bias, precision, linearity, or other performance measures. Keep in mind that differentiating between these three things is incredibly important.
For limit of detection experiments, it’s typically recommended to use multiple samples with relatively low analyte concentrations. Samples within the range of two- to four-fold higher than the determined limit of the blank will be more useful here.