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  • Guidance on IC50 data analysis
 

Statistical analysis

The PowerPoint presentations provided below are annotated with notes. The first addresses a number of the issues encountered when undertaking statistical analysis of IC50 data. It covers how IC50 values are determined from the dilution series, how to identify outlying results (above the seasonal norm), and how to monitor trends in IC50 data:

Powerpoint document Statistical analysis of IC50s

The second provides guidance on interpretation of raw data for NA activity and IC50 assays. It covers the following topics: determining standard virus dose; validation and interpretation of NA activity and IC50 analysis assays; examples of FL and CL data and data automation:

Powerpoint document Interpretation of raw data

Examples of both correct and incorrect curve fitting and point-to-point graphs for both sensitive and resistant isolates are provided below. The files contain graphical examples and accompanying explanatory notes to aid interpretation.

Powerpoint document Troubleshooting & Analysis of Curve Fitting

Powerpoint document Troubleshooting & Analysis of Point-to-Point IC50 determination


Frequently asked questions on IC50 data analysis

Q. What IC50 values can I expect to get?

Susceptibility to NI drugs is not absolute. There are typical ranges of IC50 values that differ between influenza subtypes and between oseltamivir and zanamivir for each subtype. Therefore this subtype and drug specific data should not be compared.

Also, values generated by fluorescence and chemiluminescence methods should not be compared. Typically, values generated by chemiluminescence are lower than those for the same virus and drug in the fluorescence test. IC50 values generated from the different assay methods should not be directly compared.

Influenza B viruses tend to have IC50 values 10-100 fold higher than influenza A viruses. This is normal for influenza B and this lower susceptibility does not appear to have a significant clinical impact.  IC50 values for influenza B viruses tend to be higher for oseltamivir than zanamivir.  IC50 values for H1N1 viruses tend to be higher for zanamivir than oseltamivir and IC50 values for H3N2 viruses tend to be higher for oseltamivir than zanamivir.


Q. How do I determine IC50?

IC50 values can be determined in two ways; using curve fitting software, or by a point to point analysis. Raw data (relative fluorescence or luminescence units) are plotted against the drug concentration.

Curve Fitting: Several curve fitting software are commercially available:  Graphpad Prism and Sigmaplot
Point to Point: This method uses in house excel templates. Raw data (relative fluorescence or luminescence units) are plotted against the drug concentration.  Guidance on how to undertake point-to-point analysis is provided in the IC50 analysis section


Q. How do I analyse my IC50 values?

Once you have tested a number of isolates of a given subtype against a drug, you can determine the ‘normal range’. This can be done in two ways: Box Plot Analysis and robust statistics using the standard deviation of the median absolute deviation of the median (SMAD). 

Further information on using robust statistics is available from AMC Technical Briefs.  An MS-Excel add-in for calculating the SMAD is available.  Usually, the minor outlier cut-off is chosen as the median + 1.65 SMAD, and the major outlier cut-off is chosen as the median +3 SMAD.    It is important to note that all calculations for box plot and robust statistics are performed on log data. Only after all calculations have been completed are the data back-transformed by taking the antilog.


Q. How do I know what is resistant?

There is no firm definition of a resistant IC50. Commonly used criteria are either:

     1. A value greater than 3SD from the mean (or median) value for the given subtype and drug
     2. A value 10-fold or greater than the mean (or median) value for the given subtype and drug

True resistant isolates with one of the currently known and characterised mutations tend to have IC50 values 100-10,000 times higher than the normal range for that subtype and therefore are instantly recognisable.  Confirmation of a resistant phenotype should be carried out by sequencing of the NA gene where possible.


Q. What should I do with intermediate results?

Intermediate results are those which fall over the minor cut off (whether using box and whisker or SMAD to monitor IC50s) and under the major outlier cut off and therefore are not strictly termed as resistant.

These viruses could potentially be mixture, containing quasi-species of sensitive and resistant virus or may have reduced susceptibility to drug. The IC50 test for such isolates should be repeated to confirm the result, and further characterised where possible, for example, sequencing of the NA gene.


Q. How should I monitor trends?

Trends from one influenza season to the next can be monitored using the box and whisker or SMAD method to determine whether the mean/median values and the minor and major cut off criteria are changing or remaining approximately constant.