Stepwise Optimization of the Procedure for Assessment of Circulating Progenitor Cells in Patients with Myocardial Infarction Yu-Xin Cui*, Tom Johnson, Andreas Baumbach, Barnaby C. Reeves, Chris A. Rogers, Gianni D. Angelini, Debbie Marsden, Paolo Madeddu* Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom Abstract Background: The number and functional activity of circulating progenitor cells (CPCs) is altered in diabetic patients. Furthermore, reduced CPC count has been shown to independently predict cardiovascular events. Validation of CPCs as a biomarker for cardiovascular risk stratification requires rigorous methodology. Before a standard operation protocol (SOP) can be designed for such a trial, a variety of technical issues have to be addressed fundamentally, which include the appropriate type of red blood cell lysis buffer, FMO or isotype controls to identify rare cell populations from background noise, optimal antibody dilutions and conditions of sample storage. We herein propose improvements in critical steps of CPC isolation, antigenic characterization and determination of functional competence for final application in a prospective investigation of CPCs as a biomarker of outcome following acute myocardial infarction. Methods and Findings: In this validation study, we refined the standard operating procedure (SOP) for flow cytometry characterisation and functional analysis of CPCs from the first 18 patients of the Progenitor Cell Response after Myocardial Infarction Study (ProMIS). ProMIS aims to verify the prognostic value of CPCs in patients with either ST elevation or non-ST elevation myocardial infarction with or without diabetes mellitus, using cardiac magnetic resonance imaging (MRI) for assessment of ventricular function as a primary endpoint. Results indicate crucial steps for SOP implementation, namely timely cell isolation after sampling, use of appropriate lysis buffer to separate blood cell types and minimize the acquisition events during flow cytometry, adoption of proper fluorophore combination and antibody titration for multiple antigenic detection and introduction of counting beads for precise quantification of functional CPC activity in migration assay. Conclusion and Significance: With systematic specification of factors influencing the enumeration of CPC by flow cytometry, the abundance and migration capacity of CPCs can be correctly assessed. Adoption of validated SOP is essential for refined comparison of patients with different comorbidities in the analysis of risk stratification. Citation: Cui Y-X, Johnson T, Baumbach A, Reeves BC, Rogers CA, et al. (2012) Stepwise Optimization of the Procedure for Assessment of Circulating Progenitor Cells in Patients with Myocardial Infarction. PLoS ONE 7(1): e30389. doi:10.1371/journal.pone.0030389 Editor: Gian Paolo Fadini, University of Padova, Medical School, Italy Received December 8, 2011; Accepted December 20, 2011; Published January 17, 2012 Copyright: 2012 Cui et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the National Institute for Health Research biomedical research unit in cardiovascular medicine, Bristol, UK (Funding reference: 2008/SS/BRU). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yx.cui@bristol.ac.uk (YXC); madeddu@yahoo.com (PM) Introduction Myocardial infarction (MI) is the irreversible necrosis of cardiac muscle following prolonged ischemia. It remains one of the most common causes of morbidity and mortality despite new treatment and management systems [1]. Circulating biomarkers have been the focus of recent research in order to improve risk assessment, diagnosis, and prognosis of cardiovascular disease. Single or combined biomarkers have been also used for prediction of functional outcome after an MI, as verified by echocardiography or cardiac magnetic resonance imaging (MRI). However, circulating biomarkers suffer a number of limitations. The introduction of cellular biomarkers may represent an important advancement in the evaluation of risk stratification, because the number and function of progenitor cells could directly and simultaneously address pathogenic and reparative mechanisms [2]. In particular, circulating progenitor cells (CPCs) have been proposed to be associated with occurrence and prognosis of MI [3]. Subsets of this heterogeneous population possess pluripotent potential and may help myocardial healing by direct participation in cardiac neovascularisation and remodelling, whereas the monocyte component is seemingly engaged in the paracrine modulation of the above phenomena [4]. Strong evidence indicates that CPCs are mobilized after an MI in response to cytokine stimulation, with the extent of mobilization being positively correlated with indices of cardiac contractility during recovery [5,6,7]. Risk factors may decrease CPC mobilization after a heart attack, but the findings of studies investigating CPC mobilization in diabetes are controversial. A negative correlation between CPC number and cardiovascular complications was found in Type 2 diabetes [8,9]. Other studies suggest that the viability and migration capacity of CPCs are impaired in diabetic PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30389