Flow cytometers have a constant flow rate. This enables flow cytometers to measure leukocyte concentrations in a determined volume by acquiring data at a fixed rate over a fixed time and is called constant volume acquisition (CVA). The volume aspirated by a FACS Calibur flow cytometer in 4 min at a high rate has a median of 163 microl (IQR 156-170) with TruCount tubes. Leukocyte concentrations of 26 healthy volunteers were measured twice on up to four occasions with a Bürker-Türk chamber, by single platform technology (SPT) with TruCount tubes and on the same data set using CVA. Total leukocyte concentrations determined by CVA correlated better with measurements in a Bürker-Türk (BT) chamber than with SPT. Concentrations determined with CVA were 1.86% higher than with BT whereas SPT data were 5.35% higher than BT (p<0.001), and 3.36% higher than CVA (p<0.001). At leukocyte concentrations <6 million/ml SPT correlated better with BT than CVA. The SPT measurement may be more variable because it depends on measurement of the number of beads aliquoted, the number of beads and leukocytes aspirated, where both BT counting and CVA measurements only depend on the number of leukocytes counted. CVA with PanLeukoGating can be established using microscopy as a reference, and is comparable to BT chamber and SPT determination. Leukocyte concentrations can be measured with CVA on flow cytometers in research and clinical settings.