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/microl) 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 absolute leucocyte enumeration are provided.