Flow Cytometry Shared Resource Facility (FCU) initiatives Single Cell Sorting and PCR Based upon strong support from a user survey, the laboratory acquired the Advalytix (Ampligrid) technology specifically designed for RT- and qRT-PCR on single cells in 2010. From the flow cytometry (sorting) perspective, cells are sorted individually onto specially treated slides (which have microwells for each cell) and then processed for qRT-PCR on a benchtop unit designed for microscope slides. The core has been performing single-cell deposition into PCR plates sporadically for a number of users and this is a significant improvement of the technique. The product line was sold to Beckman Coulter who discontinued it in 2012. To remediate this problem, I identified potential replacements for the slides (from the original price of $50-75 each to $0.50), expanding the matrix for sorting onto 384 well slides, and finding replacement sources for the custom PCR reagents designed to work with the system. Core personnel (Wallace) has been validating the accuracy of sorting (something that has not been published before) and has documented identifiable problems with BD (sic Aria sorters) and the advantages of sorting on a MoFlo with narrower side stream angle of incidence and on the iCyt with 0 angle of incidence. The PCR arm has been problematic, particularly since the sealing solution is out of production and the formula still considered propriety;Nguyen in the laboratory has explored several alternatives and has begun collaboration with a LMG postdoc, Peter Sykoa, for expert help and suggestions. It is our understanding, that as of the end of Feb 2013, we have made more progress on this project than our competitors, namely the Harvard flow cytometry core. Comparative analysis of compensation strategies in flow cytometry While users of the flow cytometry core have a broad range of expertise in this technology, we frequently have encountered situations where the design and implementation of their reagent panels is less than optimal. We realized that a paper documenting the proper strategies for selecting both compensation reagents and performing proper flow cytometric compensation for sample validation would benefit both our user base in the IRP as well as the flow cytometry community at large. We undertook a comprehensive analysis of typical compensation reagents, i.e. single color antibody controls using the same reagents in an antibody panels and expanded this to the use of CD8 and CD45 antibodies conjugated to each of the fluorochromes in the panel, and in collaboration with Spherotech Inc., the use of antigen-coated beads as compensation controls. This human-focused study also utilized a large number of antibody panels (12) that ranged from the simple (4-color), to 6- and 8-color, including panels that violated the common flow cytometric rules of antibody-fluorochrome selection, commercial panels, and the Euroflow panels used in the NIH trans-immunology imitative. Data acquisition is completed and an initial review of the results indicates there are a number of instances where the use of the same color antibodies (i.e. CD28 in the panel with use of CD28 as a compensation control) and either CD45 or CD8-matched fluorochrome controls are inappropriate. The results also show major differences in these beads among manufacturers adding to errors in selecting and setting compensation controls. Immunophenotypic analysis with dim fluorochromes 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 sucha 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. This research has been put on hold: Coulter J*, Wersto RP*, Yuan B, Wilsbach K, Gabrielson E. Cell-cycle independent cytotoxic effects of docetaxel in highly sensitive breast cancer cells. Status: Submitted. *co-first authors.
|Boheler, Kenneth R; Bhattacharya, Subarna; Kropp, Erin M et al. (2014) A human pluripotent stem cell surface N-glycoproteome resource reveals markers, extracellular epitopes, and drug targets. Stem Cell Reports 3:185-203|