Metal-Containing Polystyrene Beads as Standards for Mass Cytometry

Abstract

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.

Figure 1

SEM images for PS bead samples AA070-Tm synthesized in the presence of 1 % TmCl₃ added in the second stage with AA: 2 wt %/styrene (d = 2.1 μm, CV_d = 1.8%).

Figure 2

Distribution of mass cytometry intensity signal for a population of PS beads (AA070-Tm) prepared by 2-DisP in presence of TmCl₃ (1.0 wt%/styrene) and AA (2.0 wt%/styrene). The dashed-line indicates the sharp cutoff at ca. 10^{5 169}Tm signal intensity that this is likely due to detector saturation.

Figure 3

Distribution of mass cytometry intensity signal for a population of PS beads (AA086-Tm) prepared by 2-DisP in presence of TmCl₃ (0.1 wt%/styrene) and AA (2.0 wt%/styrene)

Figure 4

SEM images for PS bead samples AA120-Eu synthesized by 3-DisP in the presence of 0.1 % EuCl₃ added in the second stage with AA: 4 wt %/styrene (d = 2.2 μm, CV_d = 1.4%).

Figure 5

Distribution of mass cytometry signal intensity for a population of PS beads (AA120-Eu) prepared by 3-DisP in presence of EuCl₃ (0.1 wt%/styrene) and AA (4.0 wt%/styrene). CV_Eu = 14 %.

Figure 6

¹⁵³Eu ion release into the aqueous phase from colloidal suspensions of PS bead sample (0.5 % solids content of AA120-Eu, synthesized by 3-stage DisP) in three different buffer solutions. Beads contain 260 μg/L Eu ion (w/w styrene). pH 10.6 buffer solution: 200mM sodium carbonate/bicarbonate, pH 7.0 buffer solution: 10 mM ammonium acetate and pH 3.0 buffer solution: 50 mM sodium acetate. The right-hand y-axis represents the percentage of ¹⁵³Eu ion released into the aqueous phase to the number of ¹⁵³Eu-content of the beads.

Figure 7

Examples of bi-variant plots of mass cytometric results for (A) KG1a free cells stained with CD34-¹⁶⁹Tm and Ir-interchelator and (B) free AA120-Eu beads.

Figure 8

A bi-variant plot of mass cytometry results for: 100:1 mixture of cells and beads. Colored points represent the Ir- and Eu-positive events.

Figure 9

Comparison between the ¹⁹³Ir (from DNA intercalator) and ¹⁶⁹Tm (from CD34-¹⁶⁹Tm) averages of integrated ion intensities over the transient signals for KG1a cells gated alone (x-axis) and in a mixture with AA120-Eu beads (y-axis).