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).
Figure 1. Identification of leukocytes in human whole blood using violet side scatter on the Attune NxT Flow Cytometer. Leukocytes are outnumbered by red blood cells ~1,000-fold in whole blood and generally require enrichment by red blood cell lysis or gradient centrifugation prior to analysis. The rapid sample collection rates and inclusion of the Attune NxT No-Wash No-Lyse Filter Kit on the Attune NxT Flow Cytometer allow identification of leukocytes by scatter properties alone. (A) Using conventional blue 488 nm forward and side scatter does not allow resolution of leukocytes in whole blood. Backgate analysis using fluorescently labeled antibodies specific for leukocytes (pink) and red blood cells (blue) demonstrates this problem. (B) Resolution of leukocytes from red blood cells in whole blood is improved by incorporating violet 405 nm side scatter. (C) Backgate analysis using antibodies against the red blood cell marker glycophorin A and the leukocyte marker CD45 demonstrates the ease of identifying leukocytes in human whole blood, as opposed to in A and B. (D) Using both violet and blue side scatter allows identification of leukocytes in whole blood. This is corroborated by the backgate analysis done using the glycophorin A and CD45 labeling depicted in C, which demonstrates the different scatter properties of leukocytes and red blood cells when using violet side scatter. (E) When leukocytes are gated based on violet light scatter properties, the three main leukocyte cell populations in human blood are present: lymphocytes, monocytes, and granulocytes.
The second no-wash, no-lyse strategy involves the use of fluorescent probes or conjugated antibodies that are specific for markers expressed by red blood cells or leukocytes. For example, red blood cells express glycophorin A and white blood cells express CD45. The expression of these two markers is mutually exclusive, allowing clear identification of red vs. white blood cells and exclusion of any red blood cell coincident events (Figure 2A). A third approach is to use one of the Vybrant DyeCycle series of dyes to label nucleated cells, because mature red blood cells are anucleate (Figure 3A). Necessary materials and suggested workflows for both the violet scatter and fluorescent trigger approaches to the no-wash, no-lyse identification of leukocytes in human whole blood are included here.
* For use with violet side scatter approach.
† For use with antibody labeling approach.
‡ For use with Vybrant DyeCycle dye labeling approach.
Figure 2. Fluorescent conjugated antibody approach to identifying leukocytes in whole human blood using the Attune NxT Flow Cytometer. An alternative to the approach depicted in Figure 1 to identify leukocytes in human whole blood is using a fluorescent trigger. (A) CD45-expressing leukocytes can be targeted with fluorescently labeled antibodies, in this case anti-CD45 Pacific Orange conjugate. Anti–glycophorin A FITC conjugate is used to eliminate red blood cell coincident events. (B) Gating on CD45+, glycophorin A– cells allows identification of the three primary leukocyte populations in human blood (lymphocytes, monocytes, and granulocytes) using blue forward and side scatter while avoiding CD45+, glycophorin A+ coincident events. (C) Combining violet side scatter and backgating analysis demonstrates the accuracy of the fluorescent antibody approach to no-wash, no-lyse applications with human whole blood.
Figure 3. Identification of leukocytes in whole human blood using Vybrant DyeCycle dyes on the Attune NxT Flow Cytometer. The cell membrane– permeant Vybrant DyeCycle dyes label live nucleated cells, thus allowing identification of leukocytes in whole human blood. (A) Human whole blood labeled with Vybrant DyeCycle Ruby Stain to identify leukocytes by DNA staining. (B) Analysis of the blue forward and side light-scattering properties of the Vybrant DyeCycle Ruby–labeled cells demonstrates the ability of the dye to identify the three main leukocyte populations in human blood: lymphocytes, monocytes, and granulocytes. (C) Use of the Attune NxT No-Wash No-Lyse Filter Kit confirms the ability of Vybrant DyeCycle Ruby Stain to identify leukocytes in a whole blood sample.
We have developed three no-wash, no-lyse strategies for identifying leukocytes in human whole blood using the Attune NxT Flow Cytometer. Use of these fluorescent labeling or violet differential scatter strategies helps save time and reduces the incidence of artifacts introduced through sample preparation. It is possible to use any of the above discussed approaches as a stand-alone method for identifying leukocytes in human whole blood for further analysis.
For Research Use Only. Not for use in diagnostic procedures.