Modeling flow cytometry data for cancer vaccine immune monitoring

Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories

Lanthanide-containing polymer microspheres by multiple-stage dispersion polymerization for highly multiplexed bioassays

We describe the synthesis and characterization of metal-encoded polystyrene microspheres by multiple-stage dispersion polymerization with diameters on the order of 2 mum and a very narrow size distribution. Different lanthanides were loaded into these microspheres through the addition of a mixture of lanthanide salts (LnCl(3)) and excess acrylic acid (AA) or acetoacetylethyl methacrylate (AAEM) dissolved in ethanol to the reaction after about 10% conversion of styrene, that is, well after the particle nucleation stage was complete. Individual microspheres contain ca. 10(6)-10(8) chelated lanthanide ions, of either a single element or a mixture of elements. These microspheres were characterized one-by-one utilizing a novel mass cytometer with an inductively coupled plasma (ICP) ionization source and time-of-flight (TOF) mass spectrometry detection. Microspheres containing a range of different metals at different levels of concentration were synthesized to meet the requirements of binary encoding and enumeration encoding protocols. With four different metals at five levels of concentration, we could achieve a variability of 624, and the strategy we report should allow one to obtain much larger variability. To demonstrate the usefulness of element-encoded beads for highly multiplexed immunoassays, we carried out a proof-of-principle model bioassay involving conjugation of mouse IgG to the surface of La and Tm containing particles and its detection by an antimouse IgG bearing a metal-chelating polymer with Pr.Ahmed I. Abdelrahman, Sheng Dai, Stuart C. Thickett, Olga Ornatsky, Dmitry Bandura, Vladimir Baranov and Mitchell A. Winni

Metal-containing polystyrene beads as standards for mass cytometry

We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells.Ahmed I. Abdelrahman, Olga Ornatsky, Dmitry Bandura, Vladimir Baranov, Robert Kinach, Sheng Dai, Stuart C. Thickett, Scott Tanner and Mitchell A. Winni