Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry
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 subpopu...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-01-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-28352-2 |
| _version_ | 1797945924461264896 |
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| author | Lacey Johnson Pearl Lei Lauren Waters Matthew P. Padula Denese C. Marks |
| author_facet | Lacey Johnson Pearl Lei Lauren Waters Matthew P. Padula Denese C. Marks |
| author_sort | Lacey Johnson |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T21:02:44Z |
| format | Article |
| id | doaj.art-004ff84f68ed482faaa77734bd7ef9dc |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-10T21:02:44Z |
| publishDate | 2023-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-004ff84f68ed482faaa77734bd7ef9dc2023-01-22T12:10:35ZengNature PortfolioScientific Reports2045-23222023-01-0113111310.1038/s41598-023-28352-2Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometryLacey Johnson0Pearl Lei1Lauren Waters2Matthew P. Padula3Denese C. Marks4Research and Development, Australian Red Cross LifebloodResearch and Development, Australian Red Cross LifebloodResearch and Development, Australian Red Cross LifebloodSchool of Life Sciences, University of Technology SydneyResearch and Development, Australian Red Cross LifebloodAbstract 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.https://doi.org/10.1038/s41598-023-28352-2 |
| spellingShingle | Lacey Johnson Pearl Lei Lauren Waters Matthew P. Padula Denese C. Marks Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry Scientific Reports |
| title | Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry |
| title_full | Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry |
| title_fullStr | Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry |
| title_full_unstemmed | Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry |
| title_short | Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry |
| title_sort | identification of platelet subpopulations in cryopreserved platelet components using multi colour imaging flow cytometry |
| url | https://doi.org/10.1038/s41598-023-28352-2 |
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