Qivx Inc, ProQuant - A Technical Whitepaper

The Advanced Precision Profile
An efficient tool for calibrating, characterizing
and optimizing assay methods.
A whitepaper discussing the mathematical and scientific aspects
of the ProQuant software product
prepared by
Thomas M. Houts, Ph.D.
for
QIVX Inc. 357 C-1 Albion Way, Fort Collins, CO 80526

Abstract
(Read or Download the Whitepaper as PDF)

A poster discussing the technique was presented at the AACC (American Association for Clinical Chemistry) 36th annual Oak Ridge Conference in April of 2004, Reprints available on request.

Precision and accuracy are the two primary measures of the performance of a quantitative analytical test method or assay. Imprecision in concentration is rarely constant, and the precision profile, first described by Roger Ekins, is a convenient way to describe the precision of an assay throughout its working range. Other useful metrics, such as the upper and lower limits of quantification, are derived from the precision profile. While precision profiles have been used to characterize assay methods in routine use in clinical laboratories, they have rarely been applied in other settings, or during assay development. This is because published methods for calculating precision profiles generally require substantial amounts of data, or give unreliable estimates at the extremes of the assay range.

Developing a successful assay generally involves varying assay conditions (reagent concentrations, reaction times, measurement conditions, etc.); searching for the sets of conditions where the precision is adequate (optimal) over a suitable calibration range; and verifying that accuracy is maintained, even for "problematic" samples. Applying response-surface DOE techniques to assay development has been hampered by the difficulty in obtaining sufficient data for reliable estimates of precision, particularly since precision is a complex function of the concentration of the analyte in the sample. Some experimenters have tried optimizing on surrogate measures of performance, such as the separation in response between calibrators, with limited success. The precision profile would be the ideal tool for using DOE in optimizing assay conditions, provided that a useful profile could be obtained with reasonably small data sets.

A new approach is presented here, which efficiently utilizes data from calibrators, controls, (and unknowns, if present) to characterize a test method suitably for optimization. The precision profile is calculated, based on the pooled imprecision of the response replicates, and the slope of the dose-response curve throughout the calibration range. This calculation provides the relative standard deviation or %CV as a function of analyte concentration. Also provided are the upper and lower limits of quantification, based on the user-selected threshold for minimum acceptable precision. Accuracy can be directly estimated by including control samples with known target concentrations in the run. These direct measures of precision, accuracy, and range of quantification can be used for rapid assay characterization and optimization.

The power of the technique comes from pooling the variance in response units from all replicate groups in the run. This is based on the observation that, in many assay systems, the assumption of uniform variance in response units is valid. The software includes tests for the validity of the uniform variance assumption, and makes response transformation available should the assumption fail. Finally, the software provides confidence interval calculations for all reported values, thereby reducing the tendency for over-interpretation of the data.

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