TECHNICAL INNOVATION Flow Rate Calibration I: A Novel Approach for Performing Absolute Cell Counts

Background: Reports suggest that flow rate (FR) is constant on bench top flow cytometers. Therefore, if FR is constant, the volume acquired in a fixed time period will also be constant, enabling absolute leucocyte counting using flow rate calibration (FRC). Methods: FR stability was ascertained on a standard FACSCalibur by counting TruCount beads suspended in phosphate buffered saline over 120 s. Studies using two lysing solutions (FACS lysing solution and PharM Lyse) and corresponding sample lysates established a lysing solution calibration factor (CF). Absolute CD4 T-lymphocyte counts on 10 peripheral blood samples determined using FRC were compared with the predicate method TruCount/MultiTEST, incorporating MultiSET software. Linearity studies were also performed at three different flow rates. Results: A high degree of linearity over a wide range of counts (50 to >1,600 CD4 T lymphocytes/l) at all three pressures was observed. Importantly, there was no significant difference from the predicate method when appropriate lysing solution CF was used. Conclusions: Using a simple calibration procedure and incorporation of an appropriate lysing solution CF, we show that FRC can easily be performed. The technical details that underpin this novel approach for