How much small microplastics do we accumulate in our lungs?

Methods

Summary

All procedures take place considering any possibility of contamination. Work takes place in biosafety cabinets (with filtered airflow against the user) to prevent particles from the operator to contaminate the samples. All his follows a routine protocol in the Deheyn lab. 

Samples are weighted in the biosafety cabinet using a high precision balance. 

They are dried in an oven, with lid on each tube with a sample, but lose to allow evaporation.

The samples are digested using the conventional KOH tissue digestion method, by immersing the samples in KOH (20% in ultrafiltered MilliQ water). 

The samples are filtered on GF/F fiberglass filters (pore size 0.43microns). The filters are let sit to dry in individual Petri Dishes in the biosafety cabinet. 

The dried filters are images under a Nikon SMZ1800 stereoscope equipped with epifluorescence module and digital camera. This take 20s or so, which is the only time the samples are actually exposed to direct air during the process, thus limiting possible airborne contamination. 

The images are used to count the glowing microplastics and microfibers (both accounting for the total of sMPs). 

The counts are displayed as means and standard deviation per sample categories.

Challenges

We have a good routine procedure going and expect not technical challenges. The only one would be to analyze the sMPs for their infrared signature. The challenge will mostly be on whether these signatures will match any of the 50 control plastic we have. We believe to have the majorityy of the most common plastics... but still. Some plastics could have slightly different signatures, which we might not identify. No matter what, we will know they are plastics of some sort. 

Pre Analysis Plan

We will use analysis of variance probably to test the significance of the variation in the concentrations we find. Hard to tell in advance... depending on whether the differences are striking or not. We might also display some evolution through time (age of the patients where the samples come from). Seeing the data will help us identify what is best to analyze then for best display and best statistical consideration. We will not aim to "push" for extreme analytical methods if the data are not obvious to begin with. in this case, this means the data source is too varied for example... After all, we do not know all the habits the people who donated their lungs was! Adding more replicates of similar patients will address this.