Flow cytometric evaluation of platelet–leukocyte conjugate stability over time: methodological implications of sample handling and processing

Abstract

Platelet activation and subsequent aggregation is a vital component of atherothrombosis resulting in acute myocardial infarction. Therefore, quantifying platelet aggregation is a valuable measure for elucidating the pathogenesis of acute coronary syndromes (ACS). Circulating platelet-monocyte conjugates (PMC) as determined by flow cytometry (FCM) are an important measure of in vivo platelet aggregation. However, the influence of sample handling on FCM measurement of PMC is not well-studied. The changes in FCM measurement of PMC with variation in sample handling techniques were evaluated. The stability of PMC concentrations over time with changes in fixation and immunolabeling intervals was assessed. The effect of Time-to-Fix and Time-to-Stain on FCM PMC measurements was investigated in five healthy volunteers. Time-to-Fix (i.e., interval between phlebotomy and sample fixation) was performed at 3, 30, and 60 min. Time-to-Stain (i.e., time of fixed sample storage to staining) was performed at 1, 24, and 48 h. Increasing Time-to-Stain from 1 to 24 or 48 h resulted in lower PMC measures (p < 0.0001). A statistically significant difference in PMC measurement with increasing Time-to-Fix was not observed (p < 0.41). Postponement of sample staining has deleterious effects on the measurement of PMC via FCM. Delays in immunolabeling of fixed samples compromised measurement of PMC by 30% over the first 24 h. Staining/FCM should be completed within an hour of collection.

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Acknowledgements

This study was financially supported by the American Heart Association (11CRP7300003) and National Institute of Health (1P20 GM103492-05). We thank Jacob Schultz, Mallory Hatfield, and Joey Shaw for their assistance with sample acquisition and processing; Aaron Puckett (UofL Diabetes and Obesity Center Core Lab) for helping with flow cytometry; Allison Smith for editing the manuscript; Yong Siow and Tim O’Toole for intellectual guidance.

Funding

American Heart Association (11CRP7300003) and National Institutes of Health (1P20 GM103492-05).

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AS wrote the manuscript, executed the experiment, and interpreted the results. ARC helped with the experiment, analyzed the flow cytometry data, and contributed to the manuscript. PJT performed the biostatistical analysis, created the tables, and contributed to the manuscript. NSVS, BNA, and USO edited and reviewed the manuscript. ARA helped design the experiment and reviewed the manuscript. APD provided the lab resources, offered intellectual support, interpreted the data, and reviewed the manuscript.

Corresponding author

Correspondence to Ayesha Singh.

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All authors have no conflict of interest to disclose.

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Singh, A., Coulter, A.R., Trainor, P.J. et al. Flow cytometric evaluation of platelet–leukocyte conjugate stability over time: methodological implications of sample handling and processing. J Thromb Thrombolysis (2020). https://doi.org/10.1007/s11239-020-02186-5

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Keywords

  • Flow cytometry
  • Methods
  • Platelets
  • Monocytes
  • Specimen handling