Fixation on Histology

Updating Tissue Processing Protocols Using the “GREAT” Method

  

Winter is arriving again, and in some parts of the world, with winter comes snowflakes. Those snowflakes may come in the form of the real deal falling from the sky or even the decorative variety we create from cutting folded paper. The one thing all snowflakes have in common, though, is just how uncommon and varied they can be from one another. Filled with variations from laboratory to laboratory, tissue processing protocols are the snowflakes of the histology world. They are mostly made up of the same components and are all serving the same function for often the same types of tissues, but each laboratory’s protocol can often be as unique as a snowflake in terms of the overall protocol time and the time spent in each reagent step as a ratio. Some laboratories have not updated or evaluated their protocols for years and continue to use those that were set up by their predecessors, for better or worse, because they have no simple way to evaluate and update them. With the growing pressures to reduce turnaround time and improve quality, how do laboratories develop an efficient tissue processing protocol from all the snowflakes?

One simple method for laboratories to evaluate and potentially update their protocols is the GREAT method. GREAT stands for Greenlee Ratio Estimate for Average Time and can be used to update protocols based on average overall time length and reagent ratios. To arrive at these guidelines, hundreds of processing protocols were collected and analyzed. These protocols were compared based on characteristics such as the total protocol length and the protocol reagent time ratios and evaluated according to the quality feedback received about the protocols.

Overall, it was observed that the total protocol time and the ratios of the total time that tissues spent in dehydration, clearing, and infiltration as part of the protocol varied widely across laboratories. Since protocols operate within a generally acceptable range and a one-size-fits-all approach is not practical, the GREAT method provides ranges for determining the overall protocol length and ratios for determining the step length for each reagent group. Tissue thickness is a major factor in determining overall protocol length. As the surface-area-to-volume ratio changes with different tissue thicknesses, the time required for proper reagent penetration and action also changes, much like with fixation. Therefore, the GREAT method provides these ranges for overall processing time:

  • < 1.5 mm (ex. prostate cores, tiny GI) approximately < 1–2 hours
  • 1 – 2 mm (ex. GI, skin shaves) approximately 2–3 hours
  • 2 – 3 mm (ex. skin excisions, standard thickness) approximately 3–4 hours
  • 3 – 4 mm (surgical resections) approximately 4–6 hours
  • 4 – 5 mm (thick, large resections) approximately 6–8 hours
  • > 3 mm fatty tissue (breast, colon) approximately 8+ hours

It is important to note that the above processing times do not include fixation time or mechanical time for the processor used. Once the overall protocol time for the appropriate tissue thickness is established, the ratio rules provided by the GREAT method are applied to determine how much of the total time the tissues should spend in dehydration, clearing, and infiltration. Once again, this method is based on the analysis of hundreds of processing protocols. As shared in the webinar, the GREAT method provides some guidelines to follow to help make evaluating and updating protocols easier. Ultimately, laboratories may find that using this method can eliminate some of the guesswork and uncertainty of updating their protocols, allowing them to cut processing time without compromising quality as the below protocol results illustrate.

Scenario - biopsy protocol results graph

The GREAT method can also help improve the quality of results by suggesting a rebalancing of the reagent ratios. The key is to remember that the protocols put together by our predecessors are not set in stone. There are opportunities for improving turnaround time and quality using the simple guidelines if we choose.

You can learn more about the Updating Tissue Processing Protocols Using the “GREAT” Method in the webinar: Understanding and Updating Tissue Processing Protocols Using the GREAT Method, presented by Joshua Greenlee

References:

Greenlee, J., Webster, S., von Bueren, E. (2019). Unlocking the mystery of an effective tissue processing protocol: Using the Greenlee Ratio to Estimate Average Time (GREAT) method to determine estimated protocol length and reagent time ratio. https://www.sakuraus.com/getattachment/bd5b8dbd-66fd-41c5-8ec8-cdc7f0bc4a77/Unlocking-themystery-of-an-effective-tissue-proce


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04-02-2022 23:23

Hi. 


I just wanted to thank you for this blog. I totally agree that some of the older tissue processor protocol times could benefit from an update. 

I don’t quite understand how to figure out an updated ratio though. Also, labs are generally timid about attempting to make these kind of changes, as you already mentioned. 

I appreciate your idea and your way of thinking. You are a true scientist.