Tuesday, April 9, 2013

New technique for improving staining in older FFPE sections.

The small biotech where I am employed frequently works with with FFPE specimens that are at least 10 or more years old, and 20+ year old specimens are not particularly unusual. Any histologist can tell you that the performance of older FFPE blocks and sections used in IHC and nucleic acid hybridization studies gradually degrades with time. The logical assumption is that the target molecules are decaying and losing their affinity for the probes sent to detect them. Various antigen retrieval techniques and signal-boosting protocols have been used to try to get older tissues to take up stain more efficiently, but their usefulness seems limited. We have taken a different approach and have been experimenting with ways to "restore" older FFPE tissue samples so that the specimen stains just as it did when it was first fixed. The process we are developing seems to improve staining in both IHC and ISH studies. It also seems to help optimize IHC studies such that a uniform staining protocol can be used with similar sections of any age. I can't say too much about the details of the proprietary process that we are still developing, but I can show you some before and after images to give you an idea of how effective it is.

The first pair of images below simply illustrates the problem. The image on the left was taken in 1996 and shows HPV in situ analysis. The image on the right is the same tissue analyzed with the same probes in 2012. Nothing controversial here, just a the typical loss of signal strength that most scientists would predict in 16 year old tissue.

(Click on the image to enlarge)

In the following pairs of images we see serial sections taken from tissue blocks of various ages that have been used for  either IHC or ISH studies. In each pair, the image on the left shows the staining one might typically expect from an old section or a section from an old block. The image on the right is a similar section that was pretreated with our proprietary process, then subjected to the exact same analysis as the section shown on the left.

(Click on the images to enlarge)

These results are quite striking. So striking, in fact, that we are not sure that we totally believe them ourselves. Although we have had the effectiveness of this process confirmed by at least one third-party laboratory, we are currently actively seeking additional organizations with experience in IHC to help us validate this procedure.

If you are a lab that is struggling with poor staining in older slides, please contact me to see if we can enter into some kind of mutually beneficial collaboration. Ideally, we would set up a carefully controlled blind study where you send us pairs of problematic slides, we would treat one of the pair and send them back to you. We won't tell you which one we treated. You then stain and examine them as normal, and let us know if you see a distinct difference between them and also with controls kept at your location. Naturally there would be no charge for this. Ultimately we may be interested in commercializing this process, but first we need to be absolutely sure that it really works the way we think it does.

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