Application of analytical cytology to current issues in the biomedical sciences

点击翻译按钮获取中文摘要

Docetaxel is one of the most widely used and most effective anti-neoplastic drugs currently available. While the generally accepted mechanism for pharmacological activity of taxanes involves stabilization of microtubules in mitosis, this proposed mechanism cannot explain many of its clinical and pharmacological characteristics. We explored the relationship between mitosis and activity of docetaxel in breast cancer cell lines that display variable levels of sensitivity to the drug. All cell lines examined show an irreversible arrest in mitosis after treatment with levels of docetaxel (e.g., 10-100 nM) that have been commonly used to study drug mechanisms. However, much lower doses of docetaxel (e.g., 1 nM or less) can rapidly trigger cell death (with features of apoptosis) in highly sensitive breast cancer cell lines, such as HCC1806 and MDA-MB-231. Changes typical of early apoptosis (blunt-end DNA fragments and increased cell-surface exposure of annexin IV conjugates) were detected within 4 hours of exposure, even though no mitotic arrest is seen at this time, appeared in all phases of cell cycle. Monitoring cells with time-lapse photography confirms that cell death initiated by docetaxel does not require mitotic arrest, or even the transition through mitosis. These data suggest that docetaxel has multiple mechanisms for cell killing, including the generally recognized mechanism involving arrest of cells in mitosis. Importantly however, highly sensitive breast cancer cells are killed by relatively low levels of docetaxel through a mechanism that does not involve mitotic arrest and is initiated independently of cell cycle. With the continued development and commercial availability of flow analyzers able to routinely detect 8 colors or more, the selection of the appropriate antibody-fluorochrome combinations has become increasingly complex. Aside from differences in instrument configurations, assigning the brightest fluorochrome-antibody conjugate to the cellular antigen with the lowest expression, choosing combinations that lessen the need for spectral compensation, and avoiding fluorochrome combinations where the signal of bright populations spreads into detectors used to resolve dim populations creating false positive events are effective general recommendations. In our role as a flow cytometry core, we frequently have encountered users where the design and implementation of their reagent panels is less than optimal. Our experience does not appear to be unique; for example, since 2000, there have been more than 60 citations using Peridinin chlorophyll protein (PerCP) conjugated CD19 in multicolor panels to identify and quantify human B-cells. This list also includes the seminal paper documenting the value of ZAP-70 expression as a surrogate marker in chronic lymphocytic leukemia disease progression and patient survival (Crespo et al., 2003). The use of CD19-PerCP appears counter-intuitive and would seem to violate one of the primary principles for optimal antibody-fluorochrome selection: CD19 is expressed at low abundance on the cell surface with estimates of 27,000 molecules/cell, by contrast CD45 expression is >200,000 molecules/cell (Bikoue et al, 1996) and PerCP is generally considered a dim fluorochrome, with the relative intensity generally PE > APC >PE-Cy5 >PerCP ≥FITC. International guidelines by the Centers for Disease Control and Prevention (CDC) and the Clinical and Laboratory Standards Institute (formerly NCCLS) currently recommend 4-color panels consisting of CD3/CD4/CD8/CD45 and CD3/CD19/CD56/CD45 for the enumeration of CD4 and CD8 T-cells, B, and NK lymphocyte subsets from normal individuals. In this context, we postulated that using CD19-PerCP in such a 4-color panel to stain human whole blood specimens submitted to performance evaluation programs run under the auspices of the College of American Pathologists (CAP Flow Cytometry Immunophentypic Analysis Program) and CDC (Model Performance Evaluation Program for CD4+ T-cell Determinations) would provide an unbiased, accurate assessment of the realistic limits of reagent panels constructed with presumably less than optimal antibody-fluorochrome combinations. We extend these results to B-cell analysis in stabilized whole blood quality control products that mimic red blood cell (RBC) lysis and light scatter properties, originally developed for clinical CD4+ and CD8+ T-cell subset staining of fresh whole blood and the effect of proprietary reagents used to stabilize individual patient blood specimens over prolonged periods of time prior to staining and analysis. Overall, the results here substantiality expand the limited knowledge base of practical issues involved in normal human peripheral blood B-cell enumeration.