Flow cytometry data for determination of the Ki-67 proliferation index for diagnosis of myelodysplastic syndromes

Gating the Ki-67-FITC positive fractions is a critical step in the determination of the Ki-67 proliferation index of the different bone marrow cell populations of non-clonal cytopenic patients, MDS patients and other patients with myeloid malignancies. Therefore, we investigated whether using manually placed rectangular gates and predefined gating thresholds based on fluorescence intensities produced similar results as compared to placing polygonal gates.
Conclusion:
Placement of rectangular gates decreases the likelihood of including Ki-67 negative cells, as the gating threshold does not differ for the individual populations. Therefore, placing rectangular gates results in less inter-observer variability and more sensitively detection of differences between non-clonal cytopenic patients and MDS patients. Differences diminished by the use of predefined gating thresholds based on the fluorescent intensity of the Ki-67 staining. By using predefined gating thresholds, the intensity of the Ki-67 staining becomes a confounder in analyzing the Ki-67 proliferation indices and is influenced by a variety of factors. These factors include among others inter-operator variations, antibody batch variations and differences in staining intensities between the non-clonal cytopenic patient group and MDS patient group. These data enable reproduction of the staining and gating procedure for flow cytometric analysis of the Ki-67 proliferation index in non-clonal cytopenic and MDS patients. To correct for background staining by the conjugated primary antibodies, a negative control using FITC-directly conjugated to non-relevant, isotype immunoglobulins was used. To prevent staining abnormalities caused by ageing of the sample, flow cytometry was performed within 24 hours of receival of the femoral heads. To ensure sufficient cells for flow cytometric analyses, a white blood cell count of approximately 20 x 109 cells/L (quantified with Sysmex XN-9000) was used preferentially. The instrument setup was performed according to standard procedures. Verification of the optical alignment and fluidics system of the Navios™ Flow Cytometer was performed using Flow-Check™ Pro Fluorospheres (Beckman Coulter). The compensation for each fluorochrome was established using Flow-Set™ Pro Fluorospheres (Beckman Coulter) and was performed weekly.