Standard methods for isolating and detecting leukocytes (white blood cells) in human whole blood are time-consuming and often involve significant manipulation and enrichment prior to analysis. These sample preparation steps can result in alterations in cell physiology and loss of cell types of interest . Acoustic focusing cytometry, introduced with the Invitrogen Attune Flow Cytometer, allows high sample collection rates (up to 1,000 µL/min) without any loss in data resolution, thus eliminating the need for pre-acquisition enrichment and manipulation and helping to enable the detection of rare events in a timely manner.
In human whole blood, red blood cells outnumber white blood cells ~1,000-fold. This creates two hurdles in attempting to analyze whole blood samples without manipulation or enrichment: 1) collection of a sufficient number of white blood cell events for statistically meaningful data, and 2) differentiation of white blood cells from red blood cells given the high probability of coincident red blood cell events, which is clear when observing a conventional forward and side scatter profile of whole blood (Figure 1A). This application note outlines a strategy for the detection of white blood cells within whole blood using the Attune NxT Flow Cytometer.
We have developed three no-wash, no-lyse strategies for identifying leukocytes in whole human blood on the Attune NxT Flow Cytometer. One strategy exploits the difference in light-scattering properties between red blood cells and leukocytes. Red blood cells contain hemoglobin, a molecule that readily absorbs violet laser (405 nm) light, whereas leukocytes do not (Figure 1B), resulting in a unique scatter pattern when observing human whole blood in the context of blue (488 nm) and violet (405 nm) side scatter (SSC)  (see Figure 1D). Inclusion of the Attune NxT No-Wash No-Lyse Filter Kit (Cat. No. 100022776) in the Attune NxT Flow Cytometer filter configuration allows simultaneous measurement of both blue and violet side scatter and the differentiation of red blood cells and leukocytes based on light-scattering properties alone (Figure 1D). This can be validated using fluorescently labeled antibodies that label CD45-expressing leukocytes or glycophorin A–expressing red blood cells (Figure 1C).