We are currently exploring ways to analytically verify the effectiveness of homogenization of biological fluids, particularly whole blood, using tip mixing techniques as blood tends to sedimentation.
While tip mixing is widely used due to its simplicity and compatibility with automated liquid handling systems, verifying the effectiveness of such mixing—especially in complex matrices like blood—remains a challenge.
I would be very interested to hear from anyone who has experience with or knowledge of:
Analytical methods or assays that can reliably verify homogenization of blood or other (viscous) biological fluids via tip mixing.
The use of blood-mimicking mock fluids or synthetic analogs to simulate blood behavior during mixing validation, e.g. if fresh blood samples are not available.
Any standardized protocols or validation strategies you’ve found effective in this context.
Your input would be greatly appreciated, whether it’s a published method, an in-house solution, or even a conceptual approach.
Conceptual approach here: You could spike in a dye into the matrix of interest and test the mix settings, then aspirate the volume and use repeat dispense to create fractions into a 384-well plate for absorbance/fluorescence measurement on a plate reader. The lower the %CV between the fractions, the better the mixing.
If the test is long enough, you might need to find a way to account for dye diffusion in the test and control for it. If your concern is sedimentation, then fluorescent beads that behave similarly to your sedimenting cells could work too.
Will dye really work in whole blood though? Staining of cells usually needs to be done in a cell culture setting where the cells are more dilute and/or not impeded by other coagulants and proteins etc.? Depending on what stain you use (assuming an antibody or DNA dye) you’ll get a lot of background from lysed cells.
I’d say you need to do something downstream to test mixed fractions…e.g. extract and quantify nucleic acid (fluorescence, do not use nanodrop!), or FACS to check number of cells. This would be much more accurate. Depending on what your ultimate aim is with the blood, then you should do whatever workflow you have planned, that is the only way to truly know if it works well.
Dyes such as food colouring to check visual mixing only work for clear liquids. And then of course, it is totally subjective…
Yes I agree that using the intended functional readout is the ideal way to test the liquid handling parameters (if there aren’t many steps between the mixing step in question and the final readout and it’s not cost prohibitive). That conceptual approach was more to test a specific step in isolation when different steps are suspected to have an effect.
It’s true that that visual checking of dye mixing is subjective, but using a plate reader for spectroscopic measurement of said dye isn’t supposed to be.
