While it’s important to follow Clinical Laboratory Standards Institute (CLSI) guidelines when running interference testing, many lab technicians agree that this documentation can be difficult to get a good grasp on. Fortunately, we’ve simplified some of this information so it’s easier to interpret and put into practice. In this blog, we’ll focus on screening experiment tips.
The first step in interference testing is to define the clinical significance of an interfering substance. The EP7-A guidance document outlines materials that can be used to perform interference testing. When performing this test, it’s important to determine the maximum acceptable difference (Dmax), which is defined as the medically significant difference between the means of the test and control pools. There are resources that can assist you with this.
Setting a Dmax for your interference experiment means that you need a total allowable error (TAE) budget. It covers every source of error related to an assay, and setting a TAE “budget” will allow you to devote it to bias from your interference. For example, a one-half to one-fourth ratio for your TAE is considered a good budget.
When creating your test and control pools, it’s important to distinguish how much interferent goes in each. The test pool will have a high concentration of your interferent, while your control pool will have little to no interferent. The key is knowing how much interferent needs to be added to your test pool. For most potential interferents you’ll be working with, CLSI provides the recommended interferent concentrations. But if you’re working with an interferent that’s not in that documentation, a general rule of thumb is to test three-times the highest concentration of interferent currently documented.
Once you have your control and test pools sorted, it’s time to determine how many replicates of each you need to test in order to get an accurate measure of interference. You may want to consult CLSI sample size equations for this step. In order to do this, you’ll need to do a few things:
- Define the probability of falsely rejecting a true hypothesis
- Define the probability of accepting a false hypothesis
- Define the ratio of Dmax/repeatability expressed as a concentration or percentage
After you’ve completed these calculations and run your analysis on the types of drug interactions you want to study, you can determine the test results. Fortunately, CLSI keeps this relatively simple. The difference is only interpreted in terms of medical significance.