Platelet mass cytometry: Optimization of sample, reagent, and analysis parameters

Abstract

Platelets mediate key biological processes, including hemostasis, immunity, and inflammation. Although platelets are often treated as a homogeneous cell population, they are known to be heterogeneous in size, age, surface receptor expression, and response to agonist stimulation, raising the possibility that distinct platelet subsets perform specialized functions and that such subsets may be altered in disease settings. Attempts to identify platelet subsets by flow cytometry have had limited success due in part to limits on the number of probes that can be used at the same time and due to the challenges of compensating for probes that have large spectral overlap. We recently reported a method to identify platelet subsets by mass cytometry using a panel of 14 metal‐tagged antibodies directed at platelet surface markers. Here, we describe the technical considerations and best practices for platelet sample preparation, processing, and analysis by mass cytometry. Specifically, we show that anticoagulant choice alters platelet phenotype and function and that antibody cocktail storage and sample processing are critical for reproducibility. Additionally, we optimize sample density and instrument setup for maximal platelet transmission. Lastly, we demonstrate the importance of panel design and compensation and the use of clustering and dimension reduction to map platelet heterogeneity across resting and stimulated samples.

Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin

“…Given that a previous study has shown a subset of platelets that are packaged with tissue factor to facilitate coagulation, whether these same cells also go on to become procoagulant has not yet been explored although it seems likely given the shared function, that these same cells may go on to become procoagulant 46 . Undoubtedly there are further biomarkers to be described and this may be where the platform of CyTOF supersedes fluorescent flow cytometry as many more parameters can be simultaneously screened 24–26 . However, where single‐parameter platelet fluorescent flow cytometry is performed diagnostically this could be expanded to include panels of markers described here, allowing further aspects of platelet function to be examined within the clinical setting without requiring the additional complexity of CyTOF 47 …”

“…Automatic compensation was performed with VersaComp antibody capture beads and CytExpert (v2.1). Antibodies were used at optimal titers, and assays were designed according to platelet flow cytometry guidelines 26,32,33 …”

“…Advances in flow cytometry have increased the number of parameters that can be measured on each cell, allowing subset characterization 23 and when these multiparameter assays are coupled to computational analysis it can allow unprecedented insights into platelet heterogeneity. Recently, elegant mass cytometry (CyTOF) studies showed how an unbiased approach to surface marker characterization suggested the possibility of novel platelet subpopulations 24–26 . With this in mind we combined whole blood multiparameter fluorescent flow cytometry, with a variant of t‐stochastic neighborhood embedding (t‐SNE), fast Fourier transform‐accelerated interpolation‐based t‐SNE (FIt‐SNE) to examine platelet subpopulation formation 27 .…”

The Underestimated Role of Platelets in Severe Infection a Narrative Review

“…In patients with low platelet counts, the best option consists of flow cytometric assays of platelet activation markers. The use of flow cytometry has some advantages: a smaller volume of blood is needed without platelet-rich plasma preparation [77,[80][81][82]. Several flow cytometry approaches have been successfully used in patients with severe chronic immune thrombocytopenia, showing that impaired platelet function is associated with bleeding, independent of platelet count.…”

Mass‐tag barcoding for multiplexed analysis of human synaptosomes and other anuclear events

“…We and others have used conventional flow cytometry to analyze single synaptosomes, but with limited multiplexing, and challenged by simultaneous detection of multiple small events [4,5,13,14]. Recently, we adapted mass cytometry (CyTOF) to the analysis of single human synaptosomes (SynTOF) to increase multiplexing capacity many fold [3]; others have used CyTOF to investigate different anuclear particles, platelets [15,16]. Here, we sought to improve SynTOF by adapting commercially available masstag cell barcoding (MCB) reagents, expecting that more metal bound per particle might enhance detection of smaller events.…”