Circulating Progenitor Cells in Infarction Figure 4. Representative graphs showing the interference of different fluorophore-conjugated antibodies. Different PE-labelled antibodies were added to KDR-FITC, CD133-APC and CD34-PECy7 in individual samples. Triplicate tests were performed. Data are presented as geometric means with 95% confidence intervals. doi:10.1371/journal.pone.0030389.g004 buffer markedly increased the yield of the total cells and lymphocyte population. The presence of paraformaldehyde in the lysis buffer did not have an effect on the yield of monocytes by flow cytometry. Fixation during RBC lysis leaves white cell populations (e.g. CD34+ cells) in sticky blood and leads to their loss during washing and resuspending, which might explain why the fixative-free lysis buffer provides a higher yield of cells after the lyse-wash procedure [17]. Therefore, fixative-free RBC lysis buffer is definitely the best option for the quantification of rare progenitor cells in blood by flow cytometry. Although flow cytometry equipped with multiple lasers and fluorescence detectors is sensitive enough to identify rare cells in blood, variation in data characterising CPCs can be caused by various factors such as autofluorescence, cell aggregation, non- specific immunostaining of other cell types including dead cells/ debris, non-lysed erythrocytes, platelets as well as system fluctuation due to excessive speed, inadequate cleaning or blocking. We tested the reproducibility of a cell population indicated by a fluorophores- conjugated marker in the presence of other markers by repetition. Sometimes other fluorophore-conjugated antibodies can interfere with quantification of CPCs. Therefore, when a study is carried out, protocols must be designed with strict validation of antibodies, which must be used consistently. According to good laboratory practice, repetition (e.g. duplicate/triplicate tests) is ideal, yet flow cytometric data are often presented as a single test to reduce costs. When rare CPC populations are enumerated, attention must be given to potential contamination during the sample process, for instance, immunostaining with multiple fluorophores. The tech- nology of FMO controls has been reported to be necessary to accurately identify positive cells in the fully stained sample after multicolour flow cytometry [18,19]. In a study on rare CPC enumeration, this is definitely the case. Without the FMO controls, it is also difficult to set up proper gating due to the accumulation of background noise by spectral spillover from irrelevant fluorophores. This is more obvious when tandem fluorophores such as PE-Cy7 are combined. Due to the complexity inherent to CPC heterogeneity, the application of multiple antibodies is often necessary. However, the selection of different photospheres can still be tricky. Our titration data suggest that the efficiency of immunostaining using an individual rare progenitor cell marker can be highly heteroge- neous. Before antibodies are selected and combined, it is essential to perform individual dose-titration tests in order to identify the optimal strategy. Although fresh samples are ideal, researchers would like to understand whether CPCs in peripheral blood are stable over short periods of time since being able to store samples for a few hours makes a study more practicable. Therefore, we investigated the effect of blood freshness on CPC-marker expression. After 24 hours of storage at 4uC, both the percentage of lymphocytes and expression of most CPC markers (e.g. KDR, CD133 and CD117) decreased, suggesting that CPCs in peripheral blood are fragile or the antigen can be downregulated or internalized during storage. Furthermore, when white blood cells in 24-hour blood are isolated by Histopaque 1077, clotting can be observed on the porous barrier in a LeucoSep tube, which may potentially cause degradation of viable cells and deterioration of cellular functions in vitro. Therefore, when PB samples are used for the evaluation of CPCs, the gold standard should remain fresh blood, and ‘the fresher the better’ [20]. Practically, a same-working-day strategy (e.g. 8 hrs) should be applied for a study, but this operative window may remarkably reduce the possibility to recruit patients with an acute MI who can present at any time of day. Application of flow-cytometry to the CPC migration assay provides the means to understand how many and which CPCs respond to a particular cytokine. However, reproducibility could be reduced due to cell loss during multiple and tedious manipulation during preparation of samples for analysis. In order to enable robust high-throughput studies and track migration in a real-time manner, some improved techniques emerged which include OrisTM Cell Migration Assay (Platypus technologies, Madison, WI) and FluoroblokTM (BD Biosciences, Bedford, MA). These techniques would be encouraged to integrate cutting-edge high throughput migration methods with flow cytometry in future. In conclusion, although the interest in CPCs is steadily increasing, the lack of a consensus on standardised protocols remains a barrier to clinical exploitation [21,22]. In this study, we addressed some critical steps instrumental to optimal enumeration and characterisation of CPCs in cardiovascular patients. Our findings may be also applicable for other clinical trials when CPCs need to be enumerated, for instance, stroke, and chemo-/radio- therapies for cancer patients. PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30389