**3. Analytical phase and data reporting**

## **3.1. Instrument setup and standardization**

Optimal instrument setup and standardization is a prerequisite for reproducible results over time and among laboratories. For the analysis of RBCs, the forward scatter (FS) and side scatter (SS) voltages are set in logarithmic mode and voltages adjusted to bring all unstained RBCs into the middle of the plot and above any FS threshold/discriminator. For WBCs, light scatter voltages are set in linear mode at such values that all unstained leukocyte subsets including lymphocytes scatter above the FS threshold and are clearly clustered on scale. Photomultiplier tube (PMT) voltage optimization, standardization and computer-assisted spectral overlap compensation are mandatory steps for instrument standardization of multiparameter assays and can be performed using an instrument platform-based approach (BD Biosciences, Beckman Coulter) or interplatform-based approach [17–20].
