patients, whereas CPC counts are not dramatically altered [10,11,12]. Furthermore, the usefulness of CPCs for cardiovascu- lar risk stratification in diabetic versus non-diabetic patients remains undefined. We have designed an observational clinical trial, the Progenitor cell response after Myocardial Infarction Study (ProMIS), to verify the prognostic value of CPCs in post-MI patients with or without type 2 diabetes, using cardiac MRI as primary endpoint. Here, we report the introduction of a refined SOP for optimization of CPC isolation, enumeration and qualification in functional assays Results Analyses were carried out on the first patients recruited to the ProMIS study, without knowledge of the diabetic status or MI classification (i.e. ST segment elevation myocardial infarction - STEMI - or Non-ST Segment Elevation Myocardial Infarction - NSTEMI -). In order to provide reliable and reproducible data for this trial, we first designed the experimental strategy based on the literature and our previous experience. Furthermore, to fulfil the requirements of the International Conference on Harmonisation (ICH) - Good Clinical Practice (GCP), we considered all the critical issues relevant to each individual step of the SOP as shown in Figure 1. Particularly, we validated or optimized some critical technical points that have potential effects on the ultimate outcome of the trial in a sequential manner. Difference in FACS layout of lyse/wash and lyse/no-wash procedures of blood samples After immunostaining of human peripheral blood samples with cell surface markers, RBCs are often lysed in order to separate blood cell types and minimize the acquisition events during flow cytometry. We first investigated whether the simplified lyse/no- wash procedure, which reduces sample manipulation, would give improved cell yield over lyse/wash. As shown in Figure 2, from the samples subjected to lyse/wash step, lymphocyte and monocyte populations were separated nicely on the plot of FSC vs. SSC thus allowing easy gating of those cell sub-populations (Figure 2A). From the samples subjected to lyse/no-wash step, lymphocyte and monocyte populations could still be detected on the plot of FSC vs. SSC (Figure 2B); however, they were not as distinctive as the profile from the lyse/wash step. When dead cells were stained with the Fixable Viability Dye eFluorH 780, they could be better identified from the lyse/wash step (Figure 2C) than from the lyse/no-wash one (Figure 2D). However, the abundance of CD133+ cells enumerated by both methods was similar (Figure 2E & F). Furthermore, we found that the samples prepared by the lyse/no-wash approach frequently blocked the fluid system in the flow cytometer and it took much longer to acquire a given number of events. Comparison of RBC lysis buffers There are two main types of RBC lysis buffers, i.e. with or without a fixative such as paraformaldehyde. We investigated the effect of fixative in the RBC lysis buffers on cell characterization by FACS in 3 replicates from each of 3 patients (18 samples in total; 3 patients63 replicates62 lysis buffers). As shown in Figure 3, typical blood cell populations were separated well using either a fixative-containing lysis buffer (BD) or a fixative-free variant (Invitrogen). However, the distributions of the typical cell populations were not equally distinctive in the scatter plots of FSC vs. SSC. It appeared that blood cells treated with fixative-free lysis buffer spread more along the scattergram (Figure 3B) as compared with the fixative-containing one (Figure 3A). After Circulating Progenitor Cells in Infarction normalization by counting beads, samples treated with fixative- free lysis buffer yielded approximately 1.85-fold higher levels of total cells (P,0.001) and lymphocytes (P,0.001) and 1.28 fold higher levels of monocytes (P = 0.001) (Figure 3C). As shown in Figure 3D, the percentage of lymphocytes over total cells was similar between the two experimental conditions (P = 0.74), whereas the percentage of monocytes was lower in the samples using the fixative-free lysis buffer as compared with the fixative- containing lysis buffer (P = 0.001). Interference of the multiple antibodies conjugated with different fluorophores Because there is no single antigenic marker for CPC identification, multiple antibodies conjugated with a panel of fluorophores are commonly used for flow-cytometry profiling. Therefore, it is extremely important to test and eventually minimize the interference among different fluorophores. Particu- larly, we investigated whether different PE-conjugated antibodies (CXCR4, CD164, CD117 and TrkA) have an effect on the enumeration of three commonly-used progenitor cells markers, KDR-FITC, CD133-APC and CD34-PE-Cy7 in individual samples (60 samples in total; 4 patients63 replicates65 antibod- ies). As shown in Figure 4A, the presence of the selected PE conjugated antibodies appeared to have an effect on enumeration of KDR-FITC positive cells (P = 0.023; F-test). However, the presence of those PE-conjugated antibodies had no effect of the quantification of the selected cell populations expressing CD133- APC (P = 0.16; F-test) or CD34-PE-Cy7 (P = 0.70; F-test), as shown in Figure 4C and Figure 4B, respectively. Fluorescence-minus-one controls are necessary to quantify rare circulating progenitor cells CPCs are a relatively rare component of the circulating leukocyte population even after mobilization following an acute MI. The fluorescence-associated rare events can be easily shadowed by auto-fluorescence or spillover contamination from other fluorophores. Therefore, when different fluorophore-conju- gated antibodies are combined together, it is important to apply appropriate controls for background subtraction. We particularly investigated the effect of fluorescence minus one (FMO) controls and full isotype controls. As shown in Figure 5 (A, B and C), the combinations of isotype controls showed background noise on each single channel involved (i.e. PE, APC and PE-Cy7), probably due to potential non-specific binding and/or fluorophore spillover contamination. Importantly, reference to the individual FMO controls allowed defining the appropriate gating (Figure 5 D, E and F), for determination of the number of a particular cell population (Figure 5 G, H and I). When tandem fluorophores such as PE-Cy7 were employed, isotype controls showed relatively higher background noise (Figure 5C), therefore the role of a FMO control was even more relevant (Figure 5F). Titration of each individual antibody is essential before combination for CPC enumeration Before selected antibodies were applied for CPCs enumeration, we validated their dose dependence by serial titration. As shown in Figure 6, the signals obtained from some antigenic markers such as CD34, CD117 and TrkA are typically dose-dependent and reach saturation when a high dose of antibodies is used. The signal from samples stained with CD133 however reached a steep peak followed by gradual decline when antibody concentration was increased, which fits a parabolic-curve like pattern. The signal from samples stained with CD164 was blunted unless a high PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30389