The relapse call should be anchored first to the rare-event cluster: event count, CD45 and scatter position, concordance with the diagnostic leukemia-associated immunophenotype, and separation from internal CSF leukocytes. The ESCCA/ISCCA protocol also makes sample handling, cell recovery, and the positivity threshold central because CSF is typically hypocellular and fragile. For the reported flow-positive, FLT3-ITD PCR-negative samples, showing ungated plots and gate perturbation would help distinguish persistent disease from boundary-sensitive classification. Which cytometer, lasers, detectors, and optical filters produced these data?
Gate-shift sensitivity should be tested against the actual negative and reference control distributions, not an arbitrary offset. Current guidance separates the jobs of single-color controls, which establish compensation, and FMO controls, which place boundaries when spillover spread affects the channel of interest. A useful report would show the control overlays and repeat the functional assignment across a prespecified range of gate positions. Which cytometer, lasers, detectors, and optical filters produced the data?
Without the FMO and single-stain plots, gate sensitivity cannot be separated from spillover error. A current protocol uses single-stain controls for compensation and FMO controls to place boundaries in the presence of panel spread. I would test the reported assignment across a small, prespecified series of gate shifts, using the negative and reference samples as anchors. Which cytometer and detector configuration produced the data?
