Considerations for improving the protocol

Regarding the notable staining observed in the FMO controls for CD41, CD62-P, and CD62-E, we agree that detection of low-abundance EV subsets can be challenging. While a no-wash protocol can theoretically contribute to increased background signal, we minimized this risk by including stringent instrument cleaning steps before acquisition and by running unstained EV controls. The unstained EV control demonstrated minimal background fluorescence, indicating that the observed signal in FMOs is unlikely to be due to residual debris. The impact with autofluorescence is described in Problem 4 - Troubleshooting.

An unstained EV control tube was already included in each run, and care was taken to ensure that no debris was introduced after flow cytometer cleaning and setup. This control confirmed acceptable baseline fluorescence and supported the specificity of antibody-associated signals.

With regard to detergent lysis as a quality control step, we have not performed because introducing detergent lysis at this stage could alter antigen integrity and complicate downstream comparisons. This approach is consistent with previously published EV flow cytometry protocols.

Concerning platelet contamination, we agree that residual platelets may confound the identification of platelet-derived EVs. To address this, plasma samples were processed using a three-step centrifugation protocol analogous to that employed for platelet-free plasma preparation in 5-part full blood count (FBC) analysis. Platelet counts were confirmed to be at undetectable levels prior to EV analysis. Therefore, the likelihood of intact platelets being misclassified as platelet-derived EVs is minimal. Given this verification, an increase in centrifugation speed was not deemed necessary, as higher speeds may risk EV loss or aggregation.