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16.5.1. Standardization Standard solutions are the fundamental basis for the accuracy of assays. In preparing standards, it is usually necessary to make a number of assumptions, related to the purity and behavior of the standards. Standards and samples are assumed to react with assay reagents in the same way, and matrix effects are assumed absent. Standard solutions should be dilutions of a stock solution of highly puri ed material. Often, in the case of a hormone or other complex molecule, the standard is assigned a concentration or unit value based on a comparison with a master standard curve prepared from an international standard. 16.5.2. Matrix Effects 16.5.2.1. Recovery. Recovery experiments are designed to show whether a method measures all the analyte present in the sample. Interactions with matrix components (e.g., formation of complexes) can sometimes prevent a fraction of the analyte present from be detected. The recovery of a known amount of analyte from a sample matrix has long been taken to demonstrate accuracy. For this to hold true, the standard material must be pure and the matrix must not interfere with the reactions. When the experimental conditions have been set, the recovery experiments involve spiking a base matrix with analyte. Recovery (R) is calculated from the signal difference between the spiked base and the base itself, and is equal to (measured concentration-base concentration)/(added concentration). The volume of the spike should be small (< 10% of the total volume), so that the matrix is essentially unchanged by the spike. The recovery (%) can be calculated using Eq. 16.30. % Recovery Measured concentration 100 Calculated concentration 16:30

5

It is also necessary to determine whether the best adapted exchange architecture is centralized or distributed In the next chapter, we turn our attention to these questions..

Don t try to make a solution fit if the designers did not intend it to be used in the way you propose. Complex problems have many aspects (subproblems) to them and may require more than one solution. Examine one subproblem at a time, and solve it. If your solution happens to help solve another subproblem, that s serendipity. But don t expect it to happen every time. In fact, one solution may create other unforeseen problems, such as incompatibility with other products.

Recoveries at different concentrations are used to construct the recovery plot, as shown in Figure 16.7, where linearity with slope 1 demonstrates 100% recovery. Recoveries > 90% are considered acceptable for bioassays. In the same gure, expected deviations from the ideal behavior at low and high concentration are shown. 16.5.2.2. Parallelism. The parallelism experiment is used to determine whether an assay s accuracy depends on the analyte concentration in the sample used. This characteristic is tested by diluting a standard sample, and determining whether results agree after correction for the dilution. Alternatively, results can be plotted against expected values, ideally to yield a straight ( at) line in which the concentration is independent of the dilution factor. The diluent must be carefully selected to ensure a consistent matrix among the dilutions. Failure of the parallelism test may indicate extreme sensitivity of the assay to matrix composition, lack of sensitivity at the extremes of the standard curve or inaccurate standards. Nonideal behavior at low dilution (as shown in Fig. 16.8) can result from association of analyte with matrix components, association of assay reagents with

A prospective customer of mine wanted to replicate a database from a site in New Jersey to a remote site in Kansas. My company offered a perfectly satisfactory solution to implement this replication. However, he also wanted the replication software to hold the replicated data for four hours before applying the updates to the remote site. This way if a database administrator accidentally corrupted the database by, say, deleting a table, he wanted to have his worldwide user base switch its access point to the delayed copy in Kansas. At the time, our software could not do this. Even though my company offered three other products through which he could achieve his goals, he insisted that we provide the solution through the replication software. He did not want to hear about the issues associated with reinitializing the New Jersey copy of the database after switching to Kansas, nor in considering our detailed alternate proposals. In the end, after my consulting organization did a lot of custom work to our replication software, the customer did not select our product. He felt that the solution was cobbled together. It was. In the end, his chosen solution could not do the job adequately either. If he had been willing to consider a different approach, his problems could have easily and quickly been solved. Evan