Validation of a Multicolor Flow Cytometry System Combining Automated Sample Preparation and Advanced Analysis for Lymphocyte Immunophenotyping and Lymphoid Malignancy Diagnosis

Background: Flow cytometry has become a standard diagnostic technology in clinical hematology and immunology. The CLSI H62 guideline provides a validation framework for flow cytometric assays. Standardization and quality control remain persistent challenges, with significant inter-laboratory variability documented across platforms, reagents, and operator practices (Dorn-Beineke Kelleher et al., 2024). Objective: This study validates an 8-color immunophenotyping panel for lymphoid malignancy diagnosis and a 6-color TBNK lymphocyte subset panel on the Sysmex XF-1600 platform with PS-10 automated sample preparation and VenturiOne® analysis software. The XF-1600 has demonstrated reliable performance in B-CLL diagnosis with inter-laboratory harmonization (Weir et al., 2025) and measurable residual disease assessment in multiple myeloma (Salvia et al., 2024). Methods: PMT voltage optimization was performed across seven fluorescence channels using Spherotech COMPtrol beads. Accuracy was assessed by method comparison of 16 specimens against a reference laboratory (Pearson correlation, Bland-Altman analysis). Precision was evaluated per CLSI EP15-A3 (5 replicates/run, 2 runs/day, 5 days). Specimen stability was tested at 0, 4, 8, 24, 48, and 72 hours. Carryover was assessed using sequential high/low acquisitions. A 25-specimen stability study across three batches with T0/T1 paired testing confirmed batch-to-batch consistency. Results: Optimal PMT voltages were established for all channels (peak stain indices 13.1 to 142.6). Method comparison showed Pearson r > 0.97 for CD3+, CD4+, and CD8+ with mean biases below 1.0 percentage point. Precision CVs were below 3.5% for major populations at normal levels. All parameters remained stable within 5% of baseline through 72 hours. Carryover was below 0.5%. Conclusions: The Sysmex XF-1600 with integrated automated preparation and VenturiOne® analysis meets all validation requirements for clinical lymphocyte immunophenotyping and lymphoid malignancy diagnosis.