Preprocessing begins with loading samples into the well. However, due to the solid nature of FFPE samples, loading them into 96-well plates with blocks or curls can be challenging, tedious, and prone to cross-contamination. As a result, many laboratories choose to load these samples directly into individual tubes to minimize this risk. Your approach to preprocessing becomes crucial in light of this challenge. Some labs prefer manual preprocessing, while others opt for automated instruments.
The next step involves adding a deparaffinization solution, such as xylenes, mineral oil, or other organic solvents, and applying heat to dissolve the wax. Transferring these organic solvents presents unique challenges in terms of liquid handling, as they do not behave like the aqueous solutions commonly encountered in biological research. If you choose to automate this step, we will explain how to optimize the liquid handling process for safe transfers in the "Aspirating and Dispensing Organic Liquids" section.
Following this step, you would typically add an aqueous digestion solution and proteinase K to facilitate sample digestion. These digestion processes typically take several hours, and you may opt for overnight incubation. Depending on the protocol, kit, and temperature parameters employed, there may also be a decrosslinking step. This step involves elevating the temperature to reverse the formalin-induced crosslinks.
Preprocessing steps can be time-consuming and often tie up the instrument for extended incubations. Depending on your daily throughput requirements, you may need to consider alternatives. One approach is to maximize parallelization by performing preprocessing overnight and purification steps the following day. While this method is effective, it may require a larger platform and may not be the most efficient use of the instrument. Typically, labs either use a smaller instrument for preprocessing and transferring to a 96-well plate, or perform the initial steps manually. Your decision will be influenced by factors such as throughput needs, budget constraints, and available instrument space.
Before embarking on automating these steps, ensure you have a reliable manual magnetic bead-based protocol or kit that meets your specific requirements. This manual method will serve as a control for method development.
You will use this manual method as a benchmark to guide the development of each automated step. All observations and measurements will be relative to your manual control. Your goal is to reach the same performance as the manual control by teaching the robot how to manipulate the liquids and magnetic particles effectively.
If you need some tips to improve the performance of your manual methods, view this webinar for more information: "Successfully Overcoming the Challenges of Working with FFPE Samples"
The next step is to develop the liquid class for organic liquids in use. For more details on liquid class development, read "How to Build Liquid Classes". Since accuracy is not critical for dispensing organic liquids in this method, you can visually confirm that the action is occurring correctly without dripping, and estimate pipetting accuracy using another pipette.
If the kit uses mineral oil or a similar viscous organic liquid, follow these instructions:
- Use a slow aspiration and dispensing rate.
- Draw a small volume of air into the pipette before drawing up liquids to ensure complete liquid volume dispensing.
- Pre-wet the pipettes by pipetting the liquid up 2 to 3 times before drawing up the desired volume. This will improve accuracy.
- Draw extra reagent into the pipette to make sure the correct volume is dispensed.
If the kit uses xylene or a similar organic liquid, follow these instructions:
- Draw a small volume of air into the handler’s pipette before drawing up liquids to help dispense the entire liquid volume.
- After loading the pipette with liquid, draw up some air to prevent dripping. These liquids tend to have less cohesive force and may require more air to prevent dripping.
There are two different methods by which the next step can be completed. One approach is to first aspirate the dissolved paraffin, then perform lysis and digestion. Another approach is to perform the lysis and digestion without removal of the dissolved paraffin. Regardless of the approach you take, some oil may remain after dissolving the paraffin. Therefore, when you add the aqueous solution to perform lysis and digestion, be sure to gently mix the solution to prevent an emulsion from forming. This will help avoid the need for centrifugation to facilitate separation.
Tip: When adding the digestion solution and proteinase K, follow the manufacturer's instructions precisely. Be sure to add proteinase K directly to the sample to prevent it from losing activity by digesting itself, unless the manufacturer's protocol specifies otherwise.