Background

Flow cytometry is increasingly used in scientific investigations and healthcare; however, it requires a time-consuming, multi-step sample preparation procedure, which limits its use to specialized laboratories. Most whole blood sample preparation protocols for extracellular staining consist of at least three steps: using a venous blood sample collected in an anticoagulated sampling tube, 50-100 µl is pipetted into a reaction tube; as a second step, fluorescent antibodies are mixed and incubated with the blood to allow white blood cell staining; as a third step, the lysis solution is added to the reaction tube to allow for red blood cell removal; finally, an optional washing step is performed to reduce non-specific fluorescence.

Due to these multiple timed steps that need to be carried out in specialized laboratories, the use of flow cytometry is limited. It is, for example, not yet routinely used in the clinical field, even though the assessment of patient immune cells has proven helpful in the indication of potential disease activity (Brown and Wittwer, 2000). Some authors have tried to establish protocols with only one step, proposing to remove the lysis step; however, this method necessitates a dedicated flow cytometer, restricting its use(Petriz et al., 2018).

In this study, we propose a new universal method in which white blood cell staining and red blood cell lysis are carried out in a single step. The lysis solution is stable at room temperature (RT) and does not interfere with the staining owing to its composition, which comprises a substrate that is specifically cleaved by an enzyme uniquely present in RBC membranes (van Agthoven, 2007). White cells are maintained at neutral pH under isotonic conditions and are therefore not affected. The lysis solution can include low-dose formaldehyde (0.05%) to stabilize markers of interest (Bourgoin et al., 2020c).

The fine titration of the conjugates and their direct dilution in the lysis solution enable the final washing step to be eliminated while maintaining low background levels. Using the new Dried Unitized Reagent Assays (DURA) Innovations technology, the conjugates can be dried, which enables RT storage and removes the need for pipetting, thereby supporting better standardization. The blood sample may be of venous origin or from a fingerprick, allowing for less invasive sampling suitable for a point-of-care setting. This approach is possible thanks to an excess volume of lysis solution, which permits efficient RBC lysis in up to 50 µl of blood. The conjugate quantities have also been adjusted to stain white blood cells contained in 2-50 µl blood, with a small quantity of blood no longer being a limitation because 2 µl blood contains an average of 10,000 leukocytes. As a 100-cell subpopulation is usually considered statistically representative, using 2 µl of blood allows for analysis of subpopulations as low as 1%, which is sufficient for most routine applications.

Since management of patients with infections in the Emergency Department (ED) is challenging for practitioners, we developed a panel that enables rapid patient triage in the ED using flow cytometry. It has been shown that monitoring the expression of CD169 on monocytes (mCD169), CD64 on neutrophils (nCD64), and HLA-DR on monocytes (mHLA-DR) by flow cytometry can be indicative of viral or bacterial infection, or sepsis, respectively. We therefore established a panel consisting of antibodies targeting these three markers and evaluated the one-step method in subjects with infection and septic conditions by measuring the expression of the three infection-related markers (Bourgoin et al., 2019a, 2019b, 2020a, 2020b, 2020c, and 2021; Bedin et al., 2020; Michel et al., 2020).

Many other applications can be envisioned in fields where flow cytometry is routinely performed, such as measuring T, B, and NK cell proportions, detecting leukemias and lymphomas, and enumerating CD34⁺ stem cells for transplantation.