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Platelet proteomics on less than a drop of previously frozen, non-citrate plasma
Dylan Nicholas Tabang1, Kathrin Korff1, Kaitlyn Brannon1
1Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, United States.
Insights
This study introduces a new, sample-sparing platelet proteomics method using previously frozen plasma. This approach enables accessible platelet proteomic analysis, even from small sample volumes, aiding research in conditions like COVID-19.
Area of Science:
- Hematology
- Proteomics
- Immunophenotyping
Background:
- Platelet proteomics is challenging due to stringent sample requirements (citrate-treated, fresh plasma).
- Platelet information complements plasma data, especially for immune-challenged patients.
- Existing methods limit the use of banked, non-citrate plasma samples.
Purpose of the Study:
- Develop a sample-sparing, high-throughput platelet proteomics workflow.
- Validate the workflow using previously frozen, non-citrate platelet-rich plasma.
- Apply the method to a COVID-19 patient cohort.
Main Methods:
- Optimized centrifugation of whole blood and platelet-rich plasma.
- Analyzed platelet counts via flow cytometry on previously frozen plasma.
- Performed proteomic analysis on platelet and platelet-poor plasma samples from 79 patients.
Main Results:
- Previously frozen plasma is viable for platelet proteomic analysis with minimal artifacts.
- Platelet protein counts generally mirrored platelet counts, with exceptions in severe COVID-19.
- Dysregulated proteins in severe COVID-19 patients were linked to platelet activation and efferocytosis.
Conclusions:
- The developed method efficiently analyzes platelet proteomics from small volumes (0.5 μL) of non-citrate, previously frozen plasma.
- This approach significantly increases accessibility to banked plasma for platelet proteomic studies.
- The findings support the utility of this method for understanding immune system challenges, including COVID-19.
Abstract:
Platelets are blood components not regularly analysed with proteomics due to the conventional wisdom that plasma for platelet research must be citrate-treated and freshly sampled to minimize artifactual changes. Information from platelets is complementary to plasma, specifically regarding immunophenotyping of patients with challenged immune systems due to infection or inflammation. We sought to develop a sample-sparing, high-throughput-compatible platelet proteomics workflow applicable to previously frozen, non-citrate platelet-rich plasma, in contrast to the field's standard, and test its efficacy by applying it to a COVID-19 cohort. We examined centrifugation of whole blood and platelet-rich plasma and volume requirements of plasma for platelet analysis. Platelet and platelet-poor plasma samples were analysed from a cohort of 79 patients, consisting of COVID-19 negative non-ICU and ICU controls and patients with COVID-19 over time. Conventional platelet count was successfully performed using flow cytometry on previously frozen plasma, showing minimal platelet aggregation and cell debris, demonstrating viability of previously frozen plasma for platelet proteomic analysis. Protein counts in platelets mostly mirrored trends in platelet count, except in severe COVID-19 patients within three days of admission to the ICU. Proteins dysregulated in this group compared to controls were enriched in terms related to platelet activation, phagosome, and efferocytosis. This agrees with prior reports using conventional platelet proteomics methods. Such similar findings suggest that the method developed here can utilize non-citrate, previously frozen plasma down to 0.5 μL per sample. This will make platelet proteomics studies on already collected, banked plasma samples more accessible and increase biomolecular information gained.
