Microvesicles (MVs) are carriers of molecular and oncogenic signatures present in subsets of tumor cells and tumor-associated stroma, and a focus of cancer research. Although methods to detect MVs are mature, we were concerned that the buffer used could lead to false results when quantitating MVs by flow cytometry. In this work, we detected MVs by flow cytometry withthree different solutions: water, saline, and phosphate-buffered saline (PBS). The results demonstrated that PBS, when reacted with annexin V binding buffer, produced nano-sized vesicles even when there were no MVs in the sample. No similar events occurred in the saline and water groups (P < 0.01). Annexin V positive rate increased significantly when PBS was used as the buffer, compared to saline and water. These false negative results were also observed when we quantified some markers of MVs such as CD3 and CD19. A probable explanation for these findings is the production of insoluble Ca(H2PO4)(2) or Ca3PO4 from calcium in the binding buffer and phosphate in PBS. Thus, considering the osmotic pressure of water, we suggest that saline is a more suitable buffer when counting MVs by flow cytometry.