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使用多色成像流式细胞术鉴定冷冻保存血小板成分中的血小板亚群。

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry.

机构信息

Research and Development, Australian Red Cross Lifeblood, Alexandria, NSW, Australia.

School of Life Sciences, University of Technology Sydney, Sydney, NSW, Australia.

出版信息

Sci Rep. 2023 Jan 21;13(1):1221. doi: 10.1038/s41598-023-28352-2.

DOI:10.1038/s41598-023-28352-2
PMID:36681723
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9867743/
Abstract

Cryopreservation of platelets, at  - 80 °C with 5-6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV/PAC1/CD42b/CD62P) and a novel population (AnnV/PAC1/CD42b/CD62P) that did not align with the phenotype of aggregatory (AnnV/PAC1/CD42b/CD62P) or apoptotic (AnnV/PAC1/CD42b/CD62P) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.

摘要

血小板在 -80°C 下用 5-6% DMSO 冷冻保存会导致磷脂酰丝氨酸外显和形成细胞外囊泡（EVs），这可能介导其促凝功能。促凝血小板的表型特征与其他血小板亚群重叠。本研究旨在定义体外产生的血小板亚群的表型，随后确定冷冻保存成分中存在的亚群。新鲜血小板成分（每组 6 个）未刺激作为静息血小板的来源；或用凝血酶和胶原刺激生成聚集和促凝血小板的混合物；钙离子载体（A23187）生成促凝血小板；或用 ABT-737 生成凋亡血小板。用 DMSO 冷冻保存血小板成分（n=6），解冻后在解冻的血浆单位中重新悬浮。使用多色荧光抗体和染料面板通过成像流式细胞术识别亚群的特征。用 annexin-V（AnnV）、CD42b 和 PAC1 或 CD62P 的组合能够区分四个亚群。冷冻保存的血小板包含促凝血小板（AnnV/PAC1/CD42b/CD62P）和一种新型群体（AnnV/PAC1/CD42b/CD62P），该群体与聚集（AnnV/PAC1/CD42b/CD62P）或凋亡（AnnV/PAC1/CD42b/CD62P）亚群的表型不匹配。这些数据表明，冷冻保存血小板的止血潜力增强可能是由于冷冻诱导的促凝血小板的发展，并且可能存在其他亚群。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/2998f82af59e/41598_2023_28352_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/c1e8f62da01d/41598_2023_28352_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/20d3bdab2f1e/41598_2023_28352_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/d2fb6351bdf4/41598_2023_28352_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/9d4bb102b679/41598_2023_28352_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/215d8e5e7834/41598_2023_28352_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/2998f82af59e/41598_2023_28352_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/c1e8f62da01d/41598_2023_28352_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/20d3bdab2f1e/41598_2023_28352_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/d2fb6351bdf4/41598_2023_28352_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/9d4bb102b679/41598_2023_28352_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/215d8e5e7834/41598_2023_28352_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/16f3/9867743/2998f82af59e/41598_2023_28352_Fig6_HTML.jpg

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[4]
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[5]
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[6]
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本文引用的文献

[1]
Evaluating stored platelet shape change using imaging flow cytometry.

Platelets. 2023-12

[2]
Variation in activation marker expression within the platelet population - a new parameter for evaluation of platelet flow cytometry data.

Platelets. 2022-11-17

[3]
GPVI expression is linked to platelet size, age, and reactivity.

Blood Adv. 2022-7-26

[4]
Mechanisms Underlying Dichotomous Procoagulant COAT Platelet Generation-A Conceptual Review Summarizing Current Knowledge.

Int J Mol Sci. 2022-2-25

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Flow cytometry identifies an early stage of platelet apoptosis produced by agonists of the P2X1 and P2X7 receptors.

Platelets. 2022-5-19

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Blood Adv. 2022-5-10

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Vox Sang. 2022-3

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Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin.

J Thromb Haemost. 2021-7

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J Clin Lab Anal. 2021-5

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Extended storage of thawed platelets: Refrigeration supports postthaw quality for 10 days.

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