Complement Deposition and IgG Binding on Stored Red Blood Cells Are Independent of Storage Time

Abstract

Background: In the Netherlands, red blood cells (RBCs) are allowed to be stored up to 35 days at 2-6 °C in saline-adenine-glucose-mannitol (SAGM). During storage, RBCs undergo several changes that are collectively known as storage lesion. We investigated to what extent complement deposition and antibody binding occurred during RBC storage and investigated phagocytic uptake in vitro.

Methods: RBCs were stored for different lengths of time at 2-6 °C in SAGM. Complement deposition and antibody binding were assessed upon storage and after incubation with serum. M1- and M2-type macrophages were generated from blood monocytes to investigate RBC phagocytosis.

Results: No complement deposition was directly observed on stored RBCs, while incubation of RBCs with serum resulted in variable donor-dependent C3 deposition and IgG binding, both independent of storage time. Only 1-4% phagocytosis of stored RBCs by macrophages was observed.

Conclusion: RBCs are susceptible to complement deposition and antibody binding independent of storage time. Limited phagocytic uptake by macrophages was observed in vitro.

Keywords: Antibody binding; Complement activation; Phagocytosis; Red blood cells; Storage.

Fig. 1

C3 deposition on RBCs upon incubation with normal human serum. A Representative FACS histograms are shown of C3 and C4 deposition measured directly on short (2-6 days) and long stored (35-40 days) RBCs. B Representative FACS histograms are shown of C3 and C4 deposition measured on short (2-6 days) and long stored (35-40 days) RBCs upon incubation with NHS. C Percentages of C3 positive RBCs upon incubation with NHS are shown that were short (2-6 days) or long (35-40 days) stored (n = 7 RBC donors). The Wilcoxon test showed no significance. Grey: RBCs without fluorescently labelled antibody, black solid: HI-NHS, blue dashed: short storage, and red dotted: long storage.

Fig. 2

Complement deposition and IgG binding in ABO mismatch model. Representative FACS histograms are shown of A C3 and C4 deposition and B IgG binding measured on blood group AB RBCs upon incubation with O-typed NHS. Grey: no NHS, black solid: NHS (representative graph of 3 individual experiments).

Fig. 3

IgG binding on RBCs upon incubation with normal human serum. A Representative FACS histograms are shown of IgG binding on short (2-6 days) and long (35-40 days) stored RBCs upon incubation with NHS. B Percentages of IgG positive RBCs upon incubation with NHS are shown that were short (2-6 days) or long (35-40 days) stored (n = 5 RBC donors). The Wilcoxon test showed no significance. Grey: RBCs without fluorescently labelled antibody, blue dashed: short storage, and red dotted: long storage.

Fig. 4

C3 deposition and IgG binding partly coincides on RBCs upon incubation with normal human serum. A Representative FACS dot plots are shown of RBCs cross-incubated with (autologous and allogeneic) serum of the same donors. Q1 = C3 deposition, Q2 = both C3 deposition and IgG binding, Q3 = IgG binding and Q4 = no C3 deposition and no IgG binding. B Percentages of IgG-positive RBCs (black), C3-positive RBCs (white) and C3- and IgG-positive RBCs (grey) are shown (n = 5 RBC donors and serum).

Fig. 5

IgG and C3 opsonization on stored RBCs is not sufficient for phagocytic uptake by macrophages. A Wide field images and percentages phagocytosis are shown of the anti-RhD antibody (positive control) opsonized short (2-6 days) and long stored (35-40 days) RBCs (n = 2) by M1- and M2-type macrophages. Two-way Anova test and as post-test a Tukey multiple comparisons test was performed and showed significance. B Percentages phagocytosis by M1- and M2-type macrophages are shown of the untreated short (2-6 days) and long (35-40 days) stored RBCs (n = 3) and NHS-opsonized short (2-6 days) and long (35-40 days) stored RBCs (n = 3). Bars show mean ± standard deviation.