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Peripheral blood monocytes are heterogeneous and it was recently reported that three subsets can be identified based on CD14 and CD16 expression: CD14++/CD16- classical monocytes, CD14++/CD16+ intermediate monocytes and CD14+/CD16++ nonclassical monocytes. The mostly used technique to detect and determine the subtypes is multiparameter flowcytometry, however, there are some technical challenges in the determination of the subsets in flowcytometry, including pre-analytic, analytic and postanalytic aspects. This study aims at validation and optimalization the multiparameter flowcytometric determination of monocyte subsets. For this purpose, patients from the outdoor clinic were asked to participate by having an extra EDTA-anti-coagulated blood tube drawn. The stability of the blood sample was investigated by analyzing 20 blood samples directly and 1h, 3h, 4h and 24h after venipuncture. The influence of antibody clone (B73.1 and 3G8; BD Biosciences) against CD16 was investigated making use of another group of 20 blood samples. Reproducibility was tested and three different gating strategies, based on either CD14 back-gating, HLA-DR expression or CD24 and CD56 expression were applied to the flowcytometric results. Samples were analyzed with flowcytometry (FACSCanto II; BD). The relative size of classical monocyte population shows a significant decrease 24h after venipuncture. Using the HLA-DR based gating method, a decrease in the fraction of totoal monocytes was found. No significant differences were found when comparing the B73.1 and 3G8 antibody clone, however, the use of the 3G8 clone made it possible to make a better distinction between monocytes and granulocytes in the HLADR based gating method. all measurements show a good reproducibility (CV 0,8), although the intermediate monocytes show a higher level of variation. In order to make reliable and proper determinations of the different monocyte subsets, sample preparation and acquisition has to take place within 4 hours after venipuncture. the 3G8 clone is preferred for CD16 labeling and the HLA-DR based gating method is assumed to be the most accurate in identifying the monocyte subsets. Using above mentioned recommendations, the test shows a good reproducibility.

Detection and Differentiation of Different Monocyte Subsets: Optimalization of a Multiparameter Flowcytometric Assay