Flow cytometry for RBC damage and complement activation
Authors: Villalba Cristina Figueroa , Yurtsever Nalan , Paternoster Kayla , Gallipoli Patricia , Nash Beth , Bizzario Laurie , Shah Bhushan , Rinder Henry , Tormey Chris , Lee Edward 
Published: October 2024

Abstract

Introduction
Red blood cells (RBCs) express anionic phospholipids such as phosphatidylserine (PS) in their inner cell membrane layer. As RBCs age, physiochemical changes in their cell membranes, particularly exposure of PS on the outer membrane layer, lead to phagocytosis and clearance from circulation. PS in RBCs induces activation of the complement system, increasing binding of C3 to RBCs. Thus, quantifying PS-expressing (PS+) RBCs and complement deposition on RBCs (C3+) is a potential measure of RBC injury and may improve transfusion quality when used for predicting post-transfusion RBC recovery. Our goal was to develop a flow cytometry assay that quantifies PS+ and C3+RBCs and to assess how this assay correlates with the direct antiglobulin test for C3 (C3-DAT).

Methods
Using the Cytoflex S flow cytometer, we measured PS+ and C3+ RBCs using anti-annexin-PE (PS+RBC) and biotin-labeled anti-C3 with streptravidin-PE-Cy5 (C3+RBC). RBCs from healthy donors (NL-RBCs) with negative direct antiglobulin tests for C3 (C3-DAT) (n = 4) were analyzed. To assess the sensitivity of the assay and correlation with C3-DATs, complement control cells (CCC; Immucor) were used as a positive control for both PS+RBCs and C3+RBCs and diluted to different concentrations by mixing CCCs with NL-RBCs. Aliquots of diluted RBCs were analyzed blinded for C3-DAT. Differences between means for PS+RBC and C3+RBC were compared using two-sample t-test, and one-way ANOVA and Tukey’s HSD test were used to determine differences in C3+RBCs among a C3-DAT range of reactivities (0+ to 3+) in diluted CCC samples. P values less than 0.05 were statistically significant.

Results
In healthy, DAT-negative individuals, the mean positivity for PS+RBCs was 0.28% and for C3+RBCs was 0.34%, indicating that healthy individuals have very low percentages of RBCs with PS and C3 on their cell membrane surface. In CCC diluted samples, the mean C3+RBC percentage was 1.4% in negative C3-DAT samples, 5.9% in weak+ C3-DAT samples, 28.1% in 1+ C3-DAT samples, 67.2% in 2+ C3-DAT samples, and 99.0% in 3+ C3-DAT samples. One-way ANOVA revealed a statistically significant difference in C3+RBCs amongst all C3-DAT reactivities in diluted CCC samples (P&lt;0.001), and Tukey’s HSD test found a mean difference in C3+RBCs percentage of 26.7% between 0+ and 1+ samples (P&lt;0.001), 39.2% between 1+ and 2+ samples (P&lt;0.001), and 31.8% between 2+ and 3+ samples (P&lt;0.01). Undiluted CCC samples had a mean PS+RBC percentage of 7.0% and was statistically different from the PS+RBC percentage in NL-RBCs (P&lt;0.01).

Conclusion
We describe a feasible assay to measure both PS+RBCs and C3+RBCs using flow cytometry in which CCC can be used as a positive control for PS+RBCs. This assay could serve to evaluate the quality of transfused RBC units and provide insights into the role of phosphatidylserine expression and complement deposition for RBC clearance.