Precision and Accuracy of the Calibration Curve

A calibration/standard curve is the relationship between instrument response and the expected concentration of analyte. To adequately define the relationship between concentration and response, a sufficient number of standards/ calibrators should be used, typically 4. The reproducibility and accuracy of the standard curve across multiple ( 3) runs should be confirmed by calculating interassay precision (%CV) and accuracy (%recovery of observed concentration compared to expected concentration) for each standard. Generally, a well-defined and optimized calibration curve will demonstrate acceptable precision (%CV 20%) and acceptable accuracy (%recovery of 80-120%). 

Upper limit of quantification (ULOQ) is the highest concentration limit of the assay that can be reported with acceptable accuracy and precision. ULOQ is established after 3 assay runs using results from the standard curve. Generally, an ULOQ that demonstrates a %CV 20% and %recovery of 80-120% is considered acceptable. Samples with concentrations that fall at or above the ULOQ require sufficient dilution to bring concentrations into the report-able or dynamic range of the assay. Table 41.2 shows an example of an assessment of the precision and accuracy of a standard curve including the selection of LLOQ and ULOQ limits. 

TABLE 41.2 NGAL (Rat, Meso Scale Discovery ) Standard Curve Precision and Accuracy of Measured (Back-Calculated) Values versus Nominal Values 

Nominal Value STDl STD2 STD3 STD4 STD5 STD6 STD7 

Limit of detection (LOD) is the lowest concentration of an analyte that the bioanalytical procedure can reliably differentiate from background noise. There are several approaches for determining the LOD (ICH Harmonized Tripartite Guideline, 2005), but a common practice is to evaluate the variability of the analytical background response of blank samples. To estimate the LOD, run blank (e.g., assay buffer, zero calibrator) sample replicates ( 6) across one or more runs and calculate the mean background value 2 SD or 3SD to define the LOD. Although commonly used to define the sensitivity of an assay, LOD should be used with caution because the value is defined in an inherently variable region of the curve and is based upon a user-defined calculation. 

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