Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays

Abstract

Background: The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay setup. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry.Methods: Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur TM . Results: We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens.Conclusions: The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate. V C 2009 Clinical Cytometry Society

Standardizing immunophenotyping for the Human Immunology Project

“…The decrease in variability can be attributed to the stabilization of reagents against degradation, as well as to the elimination of pipetting variation and the avoidance of errors in reagent addition. Lyophilized-reagent plates have also been used as a means of standardizing stimulation reagents for functional assays 53,54 .…”

Validation of biomarkers to predict response to immunotherapy in cancer: Volume I — pre-analytical and analytical validation

“…Multiplexed reagent “cocktails” are generated comprising 8 or more fluorochrome-conjugated antibodies that recognize both cell surface and intracellular phenotyping molecules (e.g., CD3, CD4, CD56, and FoxP3) and intracellular readouts of activity (e.g., p-Akt, and p-ERK) following sample modulation with selected stimuli. The use of pre-formatted lyophilized-reagent plates (Lyoplates, BD Biosciences) can help to decrease staining variability compared with using individual liquid reagents in multiple studies in immunophenotyping [137] as well as functional assays [138]. …”

Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR‐inhibitors in peripheral human blood

“…S6 ribosomal protein (S6RP) is a downstream molecule of the mTOR‐signaling pathway and is activated through phosphorylation by the p70 ribosomal protein S6 kinase 1 (6). The application of mTOR‐inhibitors like SRL or ERL suppresses the phosphorylation of S6RP (p‐S6RP) and therefore S6RP should be an important target for a sensitive detection of SRL or ERL pharmacodynamic effects on T cell activation (7, 8). In contrast to other diagnostic tools to measure pharmacodynamic drug effects, flow cytometry affords a simultaneous detection of different surface and intracellular marker that set it to the appropriate method for multiparametric whole‐blood analysis and the ideal tool for pharmacodynamic T cell monitoring.…”