BackgroundMeasuring human monocyte subsets (CD14++CD16-, CD14++CD16+, and CD14+CD16++) and subset-specific monocyte-platelet aggregates (MPA) is vulnerable to analytical bias due to unavailability of a standardized methodology. We aimed to address this issue by focusing on the impacts of time-delayed sample processing and measurement between two commonly used anticoagulants. MethodsEthylenediaminetetraacetic acid (EDTA)- and sodium citrate (SC)-anticoagulated blood samples from 12 healthy donors were subject to either delayed (2-h delay, kept at 4 degrees C) or immediate processing (without fixation) before four-color flow cytometry (FCM) analysis. ResultsIn SC-anticoagulated samples, a 2-h delay in sample processing contributed to a significant decrease in CD14++CD16- monocyte percent and a reciprocal increase in CD14++CD16+ monocytes, as well as increases in all three subset-specific MPA. Similar slight, but non-significant changes were observed in EDTA-treated samples. In samples processed immediately and stored at 4 degrees C, delayed measurement at 0, 1, 3, and 5 h after processing led to a time-dependent decrease in CD14++CD16- monocyte percent and a reciprocal increase in CD14++CD16+ subset in SC-treated, but not in EDTA-treated, samples. Moreover, a time-dependent increase in all three subset-specific MPA was observed in SC-treated samples, which, to a lesser extent, was only observed in CD14++CD16+ MPA in EDTA-treated samples after storage at 4 degrees C for 3-5 h after processing. ConclusionsWe recommend EDTA for anticoagulation. Additionally, sample should be stored at 4 degrees C and processing and measuring should be performed within 2 h after harvest and 3 h after processing, respectively. (c) 2016 International Clinical Cytometry Society
