Proliferative and anti-apoptotic fractions in maturing hematopoietic cell lineages and their role in homeostasis of normal bone marrow

Recent developments in clinical flow cytometry allow the simultaneous assessment of proliferative and anti-apoptotic activity in the different hematopoietic cell lineages and during their maturation process. This can further advance the flow cytometric diagnosis of myeloid malignancies. In this study we established indicative reference values for the Ki-67 proliferation index and Bcl-2 anti-apoptotic index in blast cells, as well as maturing erythroid, myeloid and monocytic cells from normal bone marrow (BM). Furthermore, the cell fractions co-expressing both proliferation and anti-apoptotic markers were quantified.
Conclusion:
We present a novel application of an earlier developed assay that allows the simultaneous determination of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices in maturing hematopoietic cell populations of the BM. Their differential expression levels during the maturation process were in accordance with the demand and lifespan of these cell populations. The indicative reference values established in this study can act as a baseline for further cell biological and biomedical studies involving hematological malignancies. 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.