Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations

Spurgeon, Benjamin E. J.; Naseem, Khalid M.

doi:10.1002/cyto.b.21851

Cited by 12 publications

(13 citation statements)

References 36 publications

(88 reference statements)

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“…CyTOF is ever evolving, and future iterations should exploit its full potential. Panels are readily expandable, and they can be complemented by new techniques, like phosphoflow and barcoding, which allow the discovery and evaluation of signaling pathways and potential therapeutic targets [51–53]. Assay development is key to driving progress in platelet CyTOF, and we envisage future work unlocking breakthroughs in translational medicine.…”

Section: Discussionmentioning

confidence: 99%

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

2021

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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.

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Section: Discussionmentioning

confidence: 99%

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

2021

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“…Optimal antibody concentrations may be different for fixed versus unfixed cells and should be verified by titration. Some epitopes, such as that recognized by PAC1, may not stain after formaldehyde fixation, and P-selectin staining may be significantly reduced (Spurgeon & Naseem, 2020a).…”

Section: Fix-first Methods For Immunophenotyping Of Platelet Surface Receptorsmentioning

confidence: 99%

Immunophenotypic Analysis of Platelets by Flow Cytometry

et al. 2021

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Platelets are small but very abundant blood cells that play a key role in hemostasis, contributing to thrombus formation at sites of injury. The ability of platelets to perform this function, as well as functions in immunity and inflammation, is dependent on the presence of cell surface glycoproteins and changes in their quantity and conformation after platelet stimulation. In this article, we describe the characterization of platelet surface markers and platelet function using platelet‐specific fluorescent probes and flow cytometry. Unlike traditional platelet tests, immunophenotypic analysis of platelets by flow cytometry allows the analysis of platelet function in samples with very low platelet counts as often encountered in clinical situations. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Immunophenotyping of platelet surface receptors Alternate Protocol: Fix‐first method for immunophenotyping of platelet surface receptors Basic Protocol 2: Determination of platelet activation using P‐selectin expression and/or PAC1 binding Basic Protocol 3: Determination of procoagulant platelets using annexin V binding or antibodies specific for coagulation factor V/Va or X/Xa Support Protocol: Preparation of isolated platelets

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“…87 FC assessment of phosphatidylserine expression by annexin V binding can be of aid in diagnosing IPDs with enhanced or impaired procoagulant activity of platelets as reported in Stormorken and Scott syndrome, respectively. [87][88][89] Methodologies combining FC with microscopy (i.e., imaging FC), fluorescence labeling (i.e., multiplexed FC), or microfluidic techniques (i.e., real-time deformation cytometry, RT-DC) represent new generation tests, which seem promising for studying IPDs with high throughput and novel approaches, [90][91][92][93] but they are not well established for patient diagnosis, yet.…”

Section: Flow Cytometrymentioning

confidence: 99%

Diagnosing Inherited Platelet Disorders: Modalities and Consequences

2021

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Inherited platelet disorders (IPDs) are a group of rare conditions featured by reduced circulating platelets and/or impaired platelet function causing variable bleeding tendency. Additional hematological or non hematological features, which can be congenital or acquired, distinctively mark the clinical picture of a subgroup of patients. Recognizing an IPD is challenging, and diagnostic delay or mistakes are frequent. Despite the increasing availability of next-generation sequencing, a careful phenotyping of suspected patients—concerning the general clinical features, platelet morphology, and function—is still demanded. The cornerstones of IPD diagnosis are clinical evaluation, laboratory characterization, and genetic testing. Achieving a diagnosis of IPD is desirable for several reasons, including the possibility of tailored therapeutic strategies and individual follow-up programs. However, detailed investigations can also open complex scenarios raising ethical issues in case of IPDs predisposing to hematological malignancies. This review offers an overview of IPD diagnostic workup, from the interview with the proband to the molecular confirmation of the suspected disorder. The main implications of an IPD diagnosis are also discussed.

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Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations

Cited by 12 publications

References 36 publications

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

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

Immunophenotypic Analysis of Platelets by Flow Cytometry

Diagnosing Inherited Platelet Disorders: Modalities and Consequences

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