Mass cytometry is a revolutionary technology that allows for the simultaneous quantification of >40 different biomarkers with cellular resolution. The biomarkers are detected using metal-labeled antibodies as well as small-molecule probes of cell size, viability, and biochemical status. Barcoding is an important component of sample preparation because it reduces processing time, eliminates sample-to-sample variation, discriminates cell doublets, reduces the amount of antibody needed, and conserves sample. We developed a thiol-reactive tellurium-based barcode, TeMal. TeMal is nontoxic at working concentrations, compatible with metal-labeled antibodies, and can readily be applied to live or fixed cells, making it advantageous and complementary compared to existing barcoding reagents. We have demonstrated the utility of TeMal by barcoding microscale samples in situ to facilitate analysis of cells from an automated cell culture system using mass cytometry.

Mass cytometry (MC) measures metal isotope signals from single cells and bead samples. Since large numbers of isotopes can be employed as labels, mass cytometry is a powerful analytical technique for multiparameter cytometric assays. The calibration protocol in MC is a critical algorithm, which employs metal-encoded microbeads as an internal standard to correct the data for instrumental signal drift. The current generation of commercially available beads carries four lanthanide elements (cerium, europium, holmium, and lutetium). However, this is not sufficient to calibrate the full span of detection channels, ranging from yttrium (89 amu) to bismuth (209 amu), which are now available. To address this issue we prepared polystyrene microbeads encoded with seven elements (yttrium, indium, and bismuth in addition to the four lanthanides) by multistage dispersion polymerization for MC calibration and normalization. The bead synthesis conditions were optimized to obtain microbeads that were uniform in size and generated strong MC signal intensities at similar levels for the eight encoded isotopes. Metal ion leaching from the beads under storage and application conditions was also examined. We demonstrated that the precision of normalized MC signals in the MC detection channels was improved by employing seven-element-encoded microbeads as a standard.

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

Intraspecific variation in external and internal pigmentation is common among fishes and explained by a variety of biological and ecological factors. Blue-colored flesh in fishes is relatively rare but has been documented in some species of the sculpin, greenling, and perch families. Diet, starvation, photoprotection, and camouflage have all been suggested as proximate mechanisms driving blue flesh, but causal factors are poorly understood. We evaluated the relative importance of biological and spatial factors that could explain variation in blue coloration in 2021 lingcod (Ophiodon elongatus) captured across their range in the northeastern Pacific, from southeast Alaska to southern California. The probability of having blue flesh was highest for fish that were female, caught in shallower water, and smaller in body size. The incidence of blueness varied by region (4–25% of all fish) but was also confounded by differences in sex ratios of fish caught among regions. We analyzed the multivariate fatty acid composition of a subset of 175 fish from across the sampling range to test for differences in trophic biomarkers in blue lingcod. Lingcod fatty acid composition differed between regions and flesh colors but not between sexes. Blue-fleshed fish had lower concentrations of total fatty acids, 18:1ω-9, 16:1ω-7, 18:1ω-7, and ω-6 fatty acids, suggesting differences in energetics and energy storage in blue fish. While our data indicate potential links between diet and blue flesh in lingcod, important questions remain about the physiological mechanisms governing blueness and its biological consequences.

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