Leukocyte counting with the Nageotte hemocytometer: Use of fluorescence microscopy

Background: We describe a method for counting leukocytes in the Nageotte he mocytometer by fluorescence microscopy. Material and Methods: Leukocyte nuc lei were stained with a DNA fluorochrome. Hemoglobin interferes absorbing t he light emitted by the stained nuclei. In order to avoid this, the sample was diluted with a stromatolysing reagent and then centrifuged on a density gradient. Leukocytes were recovered in the pellet, while most of hemoglobi n and red blood cell stromata were discarded with the supernatant. At the e nd, the sample was concentrated 2 times. Results: The absolute limit of sen sitivity was 0.01 leukocytes/ml. Counting efficiency was measured and found to be 87.1% on average (range 35.7-135.5%). Conclusions: Counting is much easier and faster than with usual methods and less prone to subjective bias es. The method is also valid for components stored more than 4 weeks.