New Approaches to Evaluate the Immune and Endothelial Modulation Potential of Stored Red Blood Cell Concentrates

• Author / Creator

  Kipkeu, Betty J

• Red blood cell (RBC) transfusion is the most common treatment for anemia caused by blood loss due to hemorrhage or other pathological conditions. Despite being a life-saving therapy, RBC transfusion it is often associated with undesirable effects ranging from hemolytic and non-hemolytic transfusion reactions resulting from ABO mis-matching to infection transmission and transfusion related immune modulation (TRIM). Although the mechanisms involved in TRIM are multifactorial, previous studies have concentrated on the effect of RBC hypothermic storage lesion (HSL) on RBC quality and patient transfusion outcomes. The progressive biophysical and biochemical changes RBCs undergo during storage (HSL) has led to the controversial discussions as to whether transfusion of fresh versus old RBCs would alleviate the effects of TRIM. Recently, several retrospective studies have examined the clinical effects of sex-mismatched transfusions and the role of donor characteristics on patient post-transfusion outcomes. Although these studies suggest that donor age and/or sex influence transfusion outcomes in critically ill, there is a significant void in the in-vitro studies exploring plausible mechanisms for this effect. The overall objective of this thesis was to evaluate the role of donor age and sex on the immune and endothelial modulation potential of stored red blood cell concentrate (RCC) supernatants in-vitro. To achieve this objective, two novel bioassays to evaluate of RBC products were established. The monocyte monolayer assay (MMA) is a compatibility testing method for evaluating the clinical significance of RBC alloantibodies. Time-consuming monocyte isolation procedures and requirement for fresh monocytes have limited application of the MMA. This thesis assessed the viability and functional properties of monocytes from pooled cryopreserved buffy-coat (BC)-derived peripheral blood mononuclear cells for MMA application. Results showed comparable viability and phagocytosis ability of anti-D sensitized RBCs by pooled cryopreserved monocytes from BCs with fresh peripheral blood monocytes supporting its utility in assessing the clinical significance of RBC alloantibodies as well as the evaluation of the immune modulatory activity of stored RCCs in-vitro. In addition, the feasibility using the trans-epithelial electrical resistance (TEER) assay, a non-invasive real-time technique for monitoring cells in culture while using human umbilical vein endothelial cells (HUVECs) as a model for human endothelium was established. TEER assay showed that RBC HSL negatively impacts endothelial permeability in-vitro. Evaluation of the impact of donor age and sex on the immune modulation potential of stored RCC supernatants showed that RCC supernatants from fresh blood products (day 7) collected from blood donors ≥60-years old (-yo) enhanced the expression of pro-inflammatory cell adhesion molecules (CAMs) by HUVECs and had enhanced disruptive effect on HUVEC integrity compared to younger donors (≤30-yo). Additionally, a consistent trend towards lower pro-inflammatory activity following treatment with male ≤30-yo RCC supernatants was observed, as demonstrated by lower expression of CAMs throughout storage, lower disruptive effect on HUVEC integrity as well as reduced cytokines release by monocytes and HUVECs compared to the other three donor groups. The work presented in this thesis confirm that donor characteristics affect RBC quality parameters and influence the endothelial and immune modulation potential of RCC supernatants and, suggest that closer attention should be paid to RBC donor age and sex in immunomodulatory studies and their potential role in adverse transfusion outcomes. This thesis also presented tools that may be exploited to study mechanisms regulating permeability or activation of the endothelium as well as innate immune activation post-transfusion.

• Subjects / Keywords

  • Red Blood Cells, Red Blood Cell Concentrates, Immune Modulation

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-s47p-cn43

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.