If you work in the potency assay field long enough you will eventually run into problems with edge effects.  Sometimes they are blatantly obvious, and in some assays you might not notice them at first, but over time they show up as a bias in your potency results.

What causes edge effects?  There is not always a straight forward answer to this question and even when you know the answer it’s not always possible to prevent edge effects.

In cell based assays, edge effects are often caused by evaporation in the outer wells of the plate.  This leads to changes in salt concentrations in the cell medium, affecting cell metabolism in those wells.  They can also be caused by temperature gradients when the plate is placed in a heated incubator;  the outer wells tend to heat up faster than the inner wells.  Often, this will affect cell attachment and adhesion.

Lundholt et. al. write about an elegant solution here:  http://jbx.sagepub.com/cgi/content/abstract/8/5/566.  They investigated several different ways to prevent uneven cell-seeding of their plates and they came up with a simple solution.  They simply pre-incubated the plates at room temperature until the cells adhered, and then transferred them to the incubator.

Anther common way of preventing edge effects in 96 well plates is to use various humidity and temperature chambers.  One such example can be found here:  http://www.btc-bti.com/product_literature/stabilitychamber.htm.  There are also special gas permeable membranes that can be attached to the cell plate to help prevent evaporation.

Immunoassays can also suffer from edge effects.  Because of the shorter incubation times of these assays, evaporation is probably not the culprit, but instead they are often due light exposure and temperature gradients.  These causes can be exacerbated in assays where plates are stacked on top of each other during incubations.

Like in cell-based assays, common ways to prevent edge effects in immunoassays include temperature controlled chambers and plate seals.  In this case, cheaper plate seals can be used since gas exchange isn’t necessary in assays without cells.  Goetz et. al. discusses some of these solutions here:  http://gateway.nlm.nih.gov/MeetingAbstracts/ma?f=102268104.html

These are some of the physical causes and preventions of edge effects in potency assays.  However, in come cases, none of these help remove the bias from your assay, and your only recourse is to deal with it using statistical measures.  This will be the topic of future post.  I also want to try to convince you that waiting for these effects to show up during testing isn’t necessary, instead I will write about ways to discover the bias of edge effects during assay development.

Thanks for reading,
Dan

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