Reticuloendothelial Saturation Dictates the Development of RBC Resistance to Antibody-Mediated Clearance Following Incompatible Transfusion
Authors: Arthur Connie M , Mener Amanda , Patel Seema , Rodrigues Lilian Cataldi , Xue Carol , Nickel Robert Sheppard , Zimring James C. , Hendrickson Jeanne E , Stowell Sean R. 
Published: October 2019

Abstract
Background: Immune-mediated destruction of red blood cells (RBCs) normally occurs following incompatible transfusion, however some RBCs may remain in circulation after initial cellular clearance despite the presence of RBC specific antibodies. Recent studies suggest that antibody-induced “antigen-modulation” may in part be responsible for this cellular resistance to antibody-mediated removal following the initial phase of RBC clearance. However, the factors that dictate whether cells undergo clearance or antigen modulation immediately following incompatible RBC transfusion remain unknown. As previous studies suggest that reticuloendothelial phagocytic capacity is sometimes surpassed upon engagement and removal of antibody-opsonized cells, we hypothesized that rapid change in the phagocytic burden of reticuloendothelial cells may contribute to the development of RBC resistance following incompatible RBC transfusion.
Methods: Anti-Fy3 immunized or non-immunizedC57BL/6 recipient mice were first transfused with HOD.FVB RBCs expressing the HEL, OVA and Duffy chimeric antigen (HOD), followed by a second HOD.FVB RBC transfusion at various time intervals following the initial transfusion. Following transfusion, mice were evaluated at 10 min, 1h, 2h and 4h post-transfusion for HOD.FVB RBC survival, detectable HOD antigen, and RBC bound antibody by flow cytometric analysis. To examine the potential impact of RBC age on the sensitivity of HOD.FVB to antibody-induced clearance, HOD.FVB mice were injected with N-hydroxysulfosuccinimide (NHS) biotin 35 days prior to RBC isolation, to aid in differentiation of older from younger transfused RBCs, and then likewise transfused into anti-Fy3 immunized or non-immunizedC57BL/6 recipients and similarly evaluated for clearance, bound antibody and detectable antigen on biotin positive or negative transfused cells using flow cytometric analysis.
Results: While HOD.FVB RBCs transfused into anti-Fy3-immunized C57BL/6 recipients displayed a rapid clearance following the initial transfusion, the degree of HOD.FVB RBC clearance significantly decreased following secondary transfusions that occurred within 2 hours of the initial transfusion. However, 2 days following the initial transfusion, recipients cleared HOD.FVB RBCs at the same rate as immunized recipients not previously exposed to HOD.FVB RBCs. Examination of the HOD antigen and bound antibody demonstrated that antigen levels likewise displayed the most significant decreases during the same time period in which RBC clearance decreased. Removal of HOD RBCs during the initial clearance phase did not appear to reflect preferential clearance of older RBCs, as transfused biotinylated and non-biotinylated RBCs harvested 1 day or 35 days following biotinylation displayed equal levels of antibody-induced clearance.
Conclusion: Alterations in recipient clearance capacity following incompatible RBC exposure suggests that rapid phagocytic removal of RBCs may temporarily staturate the reticuloendothelial system immediately following RBC transfusion. However, antigen modulation appears to continue even while congestion of the reticuloendothelial system has halted RBC clearance. RBCs that escape the initial wave of clearance appear to become protected from antibody-mediated removal once the phagocytic capability of the recipient is restored, as the level of detectable antigen has dropped below that required for triggering phagocytic removal. The initial phase of clearance does not appear to reflect preferential clearance of older RBCs within the transfused units, but instead likely reflects stochastic removal of antibody bound RBCs, as RBC clearance occurred independent of RBC age.

Disclosures
No relevant conflicts of interest to declare.