Abstract
While ABO/Rh(D) red blood cells (RBC)-matched transfusions are generally considered as safe, a significant risk of alloimmunization to non-A/B blood group antigens exists; especially in chronically transfused patients. Indeed, alloimmunization to non-A/B antigens can be so severe that RBC transfusion can no longer be safely administered without the risk of a potentially deadly immune haemolytic reaction. Currently, no satisfactory solutions exist either to prevent blood group alloimmunization or to cost-effectively treat patients with severe alloimmunization. To address this problem, we have pioneered the immunocamouflage of donor RBC. The immunocamouflaged (stealth) RBC is manufactured by the covalent grafting of biologically safe polymers to RBC membrane proteins. As a result of the grafted polymer, non-A/B blood group antigens are biophysically and immunologically masked. Of particular interest is the immunocamouflage of the Rh(D) antigen which could be used to improve blood inventory and transfusion safety. The polymer-modified RBCs are morphologically normal and, in mice, exhibit normal in vivo survival at immunoprotective grafting concentration. In this chapter, we explore both the biophysical and immunological consequences of the grafted polymers, explore the conditions in which they might be appropriately used, and describe the technology necessary to manufacture functional transfusable units of these cells within the clinical setting.
Keywords
- red blood cell
- immunocamouflage
- alloimmunization
- Rh(D)
- polymer
1. Introduction
The transfusion of red blood cells (RBC) remains the most common, and best tolerated, form of tissue transplantation. Indeed, an estimated 108 million units of whole blood (~49 million litres) are collected annually worldwide for processing and eventual transfusion [1]. In spite of this massive collection effort, the need for blood constantly exceeds availability due to a combination of collection, manufacturing, storage and, most important clinically, biological (i.e., immunological) issues. The biological challenges facing successful RBC transfusions are vastly underappreciated, largely because of the long history and ubiquity of blood transfusions in modern medicine. Indeed, the RBC is an immunological complex cell with 35 major blood group systems that give rise to over 300 unique antigens capable of eliciting an immune response. Moreover, this immunological complexity is further exacerbated by the finding that the non-A/B (often referred to as
Among the non-A/B blood groups, the Rh system, and in particular Rh(D), is considered to be the most immunogenic antigen. Indeed, the Rh(D) antigen is highly immunogenic and when Rh(D)+ blood is transfused into an Rh(D)− individual, there is a 50% risk for the development of anti-Rh(D) antibodies resulting in very high risk of a haemolytic transfusion upon a second Rh(D)+ transfusion. Consequent to its immunogenicity, Rh(D) is always determined simultaneously with ABO type and constitutes the ‘±’ found alongside the ABO phenotype. Consequent to its immunogenicity, Rh(D) poses a significant challenge to blood operators since Type O Rh(D)− (O−) blood is the universal donor cell. In Euro-centric populations, 6–7% of the population is O− making the maintenance of an adequate inventory of this universal donor blood problematic but possible. Indeed, in North America and Europe, virtually all blood service providers experience a chronic shortage of Type O− blood. However, in other geographic regions, especially Asia, Rh(D)− individuals are extremely rare. Indeed, in China, only 0.1–0.4% of the population, regardless of ABO type, is Rh(D)−, making the Rh(D)− individual (especially with the increasing influx of European tourists) an at-risk patient [4]. Thus, within transfusion medicine, Rh(D) remains a significant problem in terms of both supply and its clinical risk.
Despite the immunological complexity of the RBC, simple ABO/Rh(D) matching has been, typically, considered sufficient for most acute transfusion needs. However, even when ABO/Rh(D) are appropriately matched, transfusion reactions still occur as mismatched non-A/B antigens do carry some immunological risks to a patient. While the incidence of clinically noteworthy (i.e.,
Historically, various interventions have been used in an attempt to prevent transfusion reactions arising from alloimmunization. While ABO/Rh(D) typing has been used since the 1940s, the practice of phenotyping some of the more problematic non-A/B/Rh(D) antigens is still uncommon and likely underlies the high frequency of alloimmunization in chronically transfused patients. In studies on β thalassemia, up to 20% of these chronically transfused individuals demonstrate
2. Bioengineering the red blood cell
Currently, no satisfactory solutions exist to prevent or cost-effectively treat blood group alloimmunization or to improve the inventory of Rh(D)− blood. To address these unmet needs, the covalent grafting of biocompatible polymers to donor RBC has been proposed to
Most commonly, the chemically activated polymers are covalently grafted to proteins at exposed lysine residues. As a result of the grafted polymer, donor blood group antigens are biophysically and immunologically masked while the modified RBC remaining biologically and functionally viable. To date, most studies have focused on mPEG as the polymer of choice due to its superior ability to both sterically and charge camouflage allogeneic RBC and its well-characterized, and safe, pharmacological profile. The basic chemical structure of mPEG is HO-(CH2CH2O)n-CH2CH2CH3. mPEG is of low toxicity and is US FDA approved for oral, intravenous, subcutaneous and intramuscular administration [29]. The mPEG polyether polymer is neutrally charged, available in an extraordinarily wide range of molecular weights, and is highly soluble in aqueous-based solutions making it very suitable for pharmacological use. In contrast, both the POZ (e.g., PEOZ) and HPG polymers are poorly soluble in aqueous solutions and only confer weak charge camouflage.
A large number of biological and biophysical studies have been done to characterize the effects of polymer grafting on immune recognition and
Indeed, one of the most promising prospects of RBC immunocamouflage is in both diminishing the risk of Rh(D) alloimmunization and safely increasing blood inventory during emergency situations or in circumstances where Rh(D)- blood is unavailable. As shown in Figure 3A, immune recognition and phagocytosis of anti-D (RhoGAM®; Rho(D) Immune Globulin (Human) RhoGAM Ultra-Filtered PLUS; Ortho Clinical Diagnostics)-opsonized Rh(D)+ RBC are blocked in a grafting concentration-dependent manner by the grafted mPEG polymer. Importantly, RhoGAM® is a highly purified and concentrated human-derived anti-D IgG antibody that is highly effective at RBC opsonization yielding Monocyte Index (MI) scores in the monocyte-monolayer assay (MMA) in the range of 60–100%. The RhoGAM® antibody is used clinically for the prevention of Rh immunization, including during and after pregnancy and other obstetrical conditions or incompatible transfusion of Rh-positive blood. However, RhoGAM® does not fully reflect the biological/clinical heterogeneity of anti-D alloantibodies arising in alloimmunized individuals. To assess the potential utility of mPEG-RBC in alloimmunized individuals, human-sourced anti-D alloantibodies (
Importantly, at immunologically protective grafting concentrations, the grafted polymer does not affect RBC structure or function as evidenced by normal morphology, O2 uptake and delivery, cellular deformability or ion transport [30, 33, 34, 37, 41, 52, 55]. While virtually indistinguishable from unmodified cells in most aspects, one interesting difference was noted between unmodified and mPEG-modified RBC. Consequent to the charge camouflage of the RBC, the cell:cell interactions necessary for Rouleaux formation were abrogated (Figure 4). Because the grafted polymer camouflages the charge of the cell necessary for cell:cell interaction, Rouleaux formation is inhibited resulting in attenuation of RBC sedimentation and, physiologically, decreased low-shear viscosity (Figure 4). Importantly, the decrease in low-shear viscosity may make the use of stealth RBC highly suitable for patients with diseases characterized by RBC-vaso-occlusive events (e.g., sickle cell). Importantly, RBC PEGylation inhibits both non-antibody (e.g., sickle cell self-aggregation) and antibody-mediated aggregation events and donor mPEG-RBC can serve as an efficient chain-breaker in pro-aggregation states [33].
The grafting of immunologically ‘inert’ polymers to the membrane of allogeneic RBC effectively camouflages multiple non-ABO antigens from immune recognition. The combined actions of both steric and charge camouflage underlie the ability of the grafted polymer to camouflage allogeneic blood group antigens (immunocamouflage) from the recipient’s immune system. These immunocamouflaged (i.e., stealth) RBC may be an effective tool in both preventing and treating alloimmunization in the chronically transfused patient; the transfusion of individual patients with rare blood phenotypes; emergency situation or geographic locations (e.g., China) where Rh(D)− blood is unavailable. Moreover, immunocamouflaged RBCs are inexpensively and easily manufactured in the clinical setting.
3. Manufacturing the ‘stealth RBC’
The immunocamouflaged RBC is unlikely to be a
To achieve the homogeneity necessary for a clinical mPEG-RBC unit (Figure 5), two scalable devices (Figures 6 and 7) utilizing micro-mixing chambers (alternatively Y-connectors to induce turbulence and mixing) have been designed, constructed and validated to semi-automate the RBC derivatization process and minimize the risk of contamination. Both approaches can be done aseptically using modifications of existing blood bags and sterile docking devices. The
4. Evaluating the potential clinical utility of the stealth RBC
While multiple studies have demonstrated that RBC immunocamouflage can effectively block immune recognition of multiple blood group antigens, the diversity of alloantibodies produced to a single blood group antigen by humans is staggering. Hence, a crucial step in the clinical use of the stealth RBC should be evaluating the potential efficacy of the stealth cell in the individual patient. While one might assume that standard serological testing techniques would suffice, this is not the case for a variety of reasons. Primary among these reasons, and as shown in Figure 3C, is that the laboratory serological score does not correlate significantly with the MI value or the risk of an acute transfusion reaction. Indeed, multiple studies have demonstrated that the antiglobulin test poorly reflects the amount of IgG bound and is, at best, a very weak predictor of RBC phagocytosis [60–63]. This confounding finding is actually by design. Serological testing is ‘overly’ sensitive in order to detect miniscule amounts of bound antibody to assure the appropriate typing of an individual or to detect the presence of a potentially dangerous alloantibody. Another potential complication is that a large number of commercial testing protocols employ PEG (as either a listed or unlisted ingredient) as a component of the testing reagents. The reagent PEG will cause the mPEG-RBC to segregate as ‘PEG likes PEG’ (Figure 8B). Hence, other predictors of the potential clinical utility of the stealth RBC for an individual patient are needed.
Perhaps the most definitive testing approach for the potential clinical value of the stealth RBC is the monocyte-monolayer assay (MMA). The MMA assesses FcγR-mediated adherence and phagocytosis of alloantibody-opsonized donor RBC by monocytes and has been clinically correlated with
Schematically, the MMA is described in Figure 9 (and experimentally demonstrated in Figure 3). The MMA examines FcγR-mediated phagocytosis
By using the MMA, the potential efficacy of RBC PEGylation can be done on an individualized basis. The probable success of the stealth RBC transfusion can be further enhanced by serologically selecting (or even better MMA testing) the best possible matches from the donor RBC inventory so as to minimize the risks of additional complications. Once the serological or MMA testing of the donor blood unit has been done, the unit can be PEGylated as described then tested against the individual’s alloantibodies via the MMA prior to transfusion into the recipient. However, the identification of the donor unit(s), PEGylation and MMA testing does require 48–96 h lead time. Hence, identification of potential patients should be done as early as possible to assure the availability to source appropriate polymer stock, prepare the PEGylation device and identify and test possible donor units. Concurrent with the evaluation of the potential clinical value of the stealth RBC, the physician/transfusion service must also receive institutional and governmental approval for their use.
5. Institutional and governmental approval for patient use
Prior to the actual clinical use of the stealth RBC in a seriously ill patient, compassionate use approval must be obtained from both the hospital Research Ethics Board (REB; or equivalent) and the appropriate governmental agencies (e.g., in Canada, Health Canada). This is likely to be a physician-driven process done in conjunction with the hospital's transfusion service and/or blood provider. Key to these requests is the need to clearly cite the lack of, or very limited availability, of suitable donor RBC. Once institutional approval has been obtained, the hospital REB would likely lead the interaction with the appropriate governmental agency (e.g., Health Canada) regarding an Investigative New Drug (IND) submission. For compassionate use in a single patient who would be likely to die in the absence of a transfusion, a formal IND submission may or may not be necessary. These steps will, obviously, change from country to country.
One question likely to be raised by the REB is whether the proposed mPEG-dosing is safe. The answer to this important question is, at least in part, addressed by recent Phase I–III clinical trials of PEGylated human haemoglobin (PEG-Hb;
6. Conclusion
Grafting of immunologically ‘inert’ polymers to the membrane of allogeneic RBC can effectively camouflage non-ABO antigens from immune recognition. These immunocamouflaged (i.e., stealth) RBCs may be an effective tool in both preventing and treating alloimmunization in the chronically transfused patient; the transfusion of individual patients with rare blood phenotypes; for emergency situation or geographic locations (e.g., China) where RhD-negative blood is unavailable. Importantly, several characteristics of the immunocamouflaged RBC may also make them highly suitable in patients/diseases characterized by RBC-mediated vaso-occlusive events (e.g., sickle cell) consequent to the polymer-mediated reduction in low-shear viscosity. For the hospital or clinic, the immunocamouflaged RBCs are inexpensively and easily manufactured using commonly available equipment and existing blood bags. Moreover, the potential clinical utility of the stealth RBC can be evaluated for the individual patient using the clinically validated monocyte-monolayer assay in which antigen-mismatched RBCs are PEGylated and then opsonized with the patient’s own alloantibody.
Acknowledgments
The authors would like to thank the past laboratory members whose work as graduate students and postdoctoral researchers have been cited in this chapter: Dr Duncheng Wang, Dr Yevgeniya Le, Dr Dana Kyluik-Price, Dr Li Li, Dr Amanda Bradley, Dr Audrey Chen and Dr Kari Murad. This study was supported by grants from the Canadian Institutes of Health Research (Grant No. 123317; MDS), Canadian Blood Services (MDS) and Health Canada (MDS). The views expressed herein do not necessarily represent the view of the federal government of Canada. We thank the Canada Foundation for Innovation and the Michael Smith Foundation for Health Research for infrastructure funding at the University of British Columbia Centre for Blood Research. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
References
- 1.
Klein HG, Spahn DR, Carson JL. Red blood cell transfusion in clinical practice. Lancet. 2007; 370 :415-426 - 2.
Reid ME, Lomas-Francis C. The Blood Group Antigen Facts Book. San Diego: Academic Press; 2003 - 3.
Mourant AE, Kopec AC, Domaniewski-Sobczak K. Blood Groups and Disease. London: Oxford University Press; 1978 - 4.
Lan JC, Chen Q, Wu DL, Ding H, Pong DB, Zhao T. Genetic polymorphism of RhD-negative associated haplotypes in the Chinese. Journal of Human Genetics. 2000; 45 :2 24-227 - 5.
NIHCC. Perioperative red cell transfusion. Journal of the American Medical Association. 1988; 260 :2700-2703 - 6.
Fluit CR, Kunst VA, Drenthe-Schonk AM. Incidence of red cell antibodies after multiple blood transfusion. Transfusion. 1990; 30 :532-535 - 7.
Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams A, Lubin B. Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. New England Journal of Medicine. 1990; 322 :1617-1621 - 8.
Martin S. Fundamentals of Immunology for Blood Bankers. In: Harmening DM, editor. Modern Blood Banking and Transfusion Practices. Philadelphia, PA. F.A. Davis Company; 1994. p. 43-68. - 9.
Moore SB, Taswell HF, Pineda AA, Sonnenberg CL. Delayed hemolytic transfusion reaction. American Society of Clinical Pathologists. 1979; 74 :94-97 - 10.
Kennedy MS, Julius C. Transfusion Therapy. In: Harmening DM, editor. Philadelphia, PA. F.A. Davis Company; 1994. pp. 316-333. - 11.
Economidou J, Constantoulakis M, Augoustaki O, Adinolfi M. Frequency of antibodies to various antigenic determinants in polytransferred patients with homozygous thalassaaemia in Greece. Vox Sanguinis. 1971; 20 :252-258 - 12.
Luban NL. Variability in rates of alloimmunization in different groups of children with sickle cell disease: Effect of ethnic background. American Journal of Pediatric Hematology-Oncology. 1989; 11 :314-319 - 13.
Valeri CR, Ragno G, Van Houten P, Rose L, Rose M, Egozy Y, Popovsky MA. Automation of the glycerolization of red blood cells with the high-separation bowl in the Haemonetics ACP 215 instrument. Transfusion. 2005; 45 :1621-1627 - 14.
Valeri CR. Status report on the quality of liquid and frozen red blood cells. Vox Sanguinis. 2002; 83 (Suppl 1):193-196 - 15.
Rosenblatt MS, Hirsch EF, Valeri CR. Frozen red blood cells in combat casualty care: Clinical and logistical considerations. Military Medicine. 1994; 159 :392-397 - 16.
Skrabut EM, Crowley JP, Catsimpoolas N, Valeri CR. The effect of cryogenic storage on human erythrocyte membrane proteins as determined by polyacrylamide-gel electrophoresis. Cryobiology. 1976; 13 :395-403 - 17.
Valeri CR, Runck AH. Viability of glycerolized red blood cells frozen in liquid nitrogen. Transfusion. 1969; 9 :306-313 - 18.
Valeri CR, Runck AH. Long term frozen storage of human red blood cells: Studies in vivo and in vitro of autologous red blood cells preserved up to six years with high concentrations of glycerol. Transfusion. 1969; 9 :5-14 - 19.
Valeri CR, Brodine CE. Current methods for processing frozen red cells. Cryobiology. 1968; 5 :129-135 - 20.
Beattie KM, Shafer AW. Broadening the base of a rare donor program by targeting minority populations. Transfusion. 1986; 26 :401-404 - 21.
McPherson ME, Anderson AR, Haight AE, Jessup P, Castillejo MI, Hillyer CD, Josephson CD. Transfusion management of sickle cell patients during bone marrow transplantation with matched sibling donor. Transfusion. 2009; 49 :1977-1986 - 22.
Anstee DJ. Red cell genotyping and the future of pretransfusion testing. Blood. 2009; 114 :248-256 - 23.
Osby M, Shulman IA. Phenotype matching of donor red blood cell units for nonalloimmunized sickle cell disease patients: A survey of 1182 North American laboratories. Archives of Pathology & Laboratory Medicine. 2005; 129 :190-193 - 24.
Castro O, Sandler SG, Houston-Yu P, Rana S. Predicting the effect of transfusing only phenotype-matched RBCs to patients with sickle cell disease: Theoretical and practical implications. Transfusion. 2002; 42 :684-690 - 25.
Vichinsky EP, Luban NL, Wright E, Olivieri N, Driscoll C, Pegelow CH, Adams RJ. Prospective RBC phenotype matching in a stroke-prevention trial in sickle cell anemia: A multicenter transfusion trial. Transfusion. 2001; 41 :1086-1092 - 26.
Rosse WF, et al. Transfusion and alloimmunization in sickle cell disease. Blood. 1990; 76 :1431-1437 - 27.
Ambruso DR, Githens JH, Alcorn R, Dixon DJ, Brown LJ, Vaugn WM, Hays T. Experience with donors matched for minor blood group antigens in patients with sickle cell anemia who are receiving chronic transfusion therapy. Transfusion. 1987; 27 :94-98 - 28.
Kyluik-Price DL, Li L, Scott MD. Comparative efficacy of blood cell immunocamouflage by membrane grafting of methoxypoly(ethylene glycol) and polyethyloxazoline. Biomaterials. 2014; 35 :412-422 - 29.
Fruijtier-Polloth C. Safety assessment on polyethylene glycols (PEGs) and their derivatives as used in cosmetic products. Toxicology. 2005; 214 :1-38 - 30.
Scott MD, Murad KL, Koumpouras F, Talbot M, Eaton JW. Chemical camouflage of antigenic determinants: Stealth erythrocytes. Proceedings of the National Academy of Sciences of the United States of America. 1997; 94 :7566-7571 - 31.
Scott MD, Murad KL. Cellular camouflage: Fooling the immune system with polymers. Current Pharmaceutical Design. 1998; 4 :423-438 - 32.
Murad KL, Gosselin EJ, Eaton JW, Scott MD. Stealth cells: Prevention of major histocompatibility complex class II-mediated T-cell activation by cell surface modification. Blood. 1999; 94 :2135-2141 - 33.
Murad KL, Mahany KL, Brugnara C, Kuypers FA, Eaton JW, Scott MD. Structural and functional consequences of antigenic modulation of red blood cells with methoxy poly(ethylene glycol). Blood. 1999; 93 :2121-2127 - 34.
Scott MD, Bradley AJ, Murad KL. Camouflaged blood cells: Low-technology bioengineering for transfusion medicine? Transfusion Medicine Reviews. 2000; 14 :53-63 - 35.
Bradley AJ, Test ST, Murad KL, Mitsuyoshi J, Scott MD. Interactions of IgM ABO antibodies and complement with methoxy-PEG-modified human RBCs. Transfusion. 2001; 41 :1225-1233 - 36.
Chen AM, Scott MD. Current and future applications of immunological attenuation via pegylation of cells and tissue. BioDrugs. 2001; 15 :833-847 - 37.
Bradley AJ, Murad KL, Regan KL, Scott MD. Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: Size does matter. Biochimica et Biophysica Acta. 2002; 1561 :147-158 - 38.
Chen AM, Scott MD. Immunocamouflage: Prevention of transfusion-induced graft-versus-host disease via polymer grafting of donor cells. Journal of Biomedical Materials Research: Part A. 2003; 67 :626-636 - 39.
Scott MD, Bradley AJ, Murad KL. Stealth erythrocytes: Effects of polymer grafting on biophysical, biological and immunological parameters. Blood Transfusion. 2003; 1 :244-265 - 40.
Scott MD, Chen AM. Beyond the red cell: Pegylation of other blood cells and tissues. Transfusion Clinique et Biologique. 2004; 11 :40-46 - 41.
Bradley AJ, Scott MD. Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 2004; 807 :163-168 - 42.
McCoy LL, Scott MD. Broad spectrum antiviral prophylaxis: Inhibition of viral infection by polymer grafting with methoxypoly(ethylene glycol). In: Torrence PF, editor. Antiviral Drug Discovery for Emerging Diseases and Bioterrorism Threats. Hoboken, NJ: Wiley & Sons; 2005. pp. 379-395 - 43.
Chen AM, Scott MD. Comparative analysis of polymer and linker chemistries on the efficacy of immunocamouflage of murine leukocytes. Artificial Cells, Blood Substitutes, and Immobilization Biotechnology. 2006; 34 :305-322 - 44.
Scott MD. Inactivation of prion proteins via covalent grafting with methoxypoly(ethylene glycol). Medical Hypotheses. 2006; 66 :387-393 - 45.
Bradley AJ, Scott MD. Immune complex binding by immunocamouflaged [poly(ethylene glycol)-grafted] erythrocytes. American Journal of Hematology. 2007; 82 :970-975 - 46.
Sutton TC, Scott MD. The effect of grafted methoxypoly(ethylene glycol) chain length on the inhibition of respiratory syncytial virus (RSV) infection and proliferation. Biomaterials. 2010; 31 :4223-4230 - 47.
Le Y, Scott MD. Immunocamouflage: The biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) [mpeg]. Acta Biomaterialia. 2010; 6 :2631-2641 - 48.
Rossi NA, Constantinescu I, Brooks DE, Scott MD, Kizhakkedathu JN. Enhanced cell surface polymer grafting in concentrated and nonreactive aqueous polymer solutions. Journal of the American Chemical Society. 2010; 132 :3423-3430 - 49.
Rossi NA, Constantinescu I, Kainthan RK, Brooks DE, Scott MD, Kizhakkedathu JN. Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols. Biomaterials. 2010; 31 :4167-4178 - 50.
Greco CA, Maurer-Spurej E, Scott MD, Kalab M, Nakane N, Ramirez-Arcos SM. PEGylation prevents bacteria-induced platelet activation and biofilm formation in platelet concentrates. Vox Sanguinis. 2011; 100 :336-339 - 51.
Kyluik DL, Sutton TC, Le Y, Scott MD. Polymer-mediated broad spectrum antiviral prophylaxis: Utility in high risk environments. In: Carpi A, editor. Progress in Molecular and Environmental Bioengineering—From Analysis and Modeling to Technology Applications. Rijeka: Intech; 2011. pp. 167-190. ISBN 978-953-307-268-5 - 52.
Wang D, Kyluik DL, Murad KL, Toyofuku WM, Scott MD. Polymer-mediated immunocamouflage of red blood cells: Effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells. Science China Life Sciences. 2011; 54 :589-598 - 53.
Wang D, Toyofuku WM, Chen AM, Scott MD. Induction of immunotolerance via mPEG grafting to allogeneic leukocytes. Biomaterials. 2011; 32 :9494-9503 - 54.
Chapanian R, Constantinescu I, Brooks DE, Scott MD, Kizhakkedathu JN. In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells. Biomaterials. 2012; 33 :3047-3057 - 55.
Wang D, Toyofuku WM, Scott MD. The potential utility of methoxypoly(ethylene Glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine. Biomaterials. 2012; 33 :3002-3012 - 56.
Wang D, Toyofuku WM, Kyluik DL, Scott MD. Use of flow cytometry in the in vitro and in vivo analysis of tolerance/anergy induction by immunocamouflage. In: Schmid I, editor. Flow Cytometry-Recent Perspectives. Rijeka, Croatia InTech; 2012. pp. 133-150 - 57.
Le Y, Toyofuku WM, Scott MD. Immunogenicity of murine mPEG-red blood cells and the risk of anti-PEG antibodies in human blood donors. Experimental Hematology. 2017; 47 :36-47 - 58.
Kyluik-Price DL, Scott MD. Effects of methoxypoly (ethylene glycol) mediated immunocamouflage on leukocyte surface marker detection, cell conjugation, activation and alloproliferation. Biomaterials. 2016; 74 :167-177 - 59.
Li L, Noumsi GT, Kwok YY, Moulds JM, Scott MD. Inhibition of phagocytic recognition of anti-D opsonized Rh D+ RBC by polymer-mediated immunocamouflage. American Journal of Hematology. 2015; 90 :1165-1170 - 60.
Branch DR, Gallagher MT, Mison AP, Sy Siok Hian AL, Petz LD. In vitro determination of red cell alloantibody significance using an assay of monocyte-macrophage interaction with sensitized erythrocytes. British Journal of Haematology. 1984; 56 :19-29 - 61.
Branch DR, Gallahger MT. Correlation of in vivo alloantibody significance or insignificance with an in vitro monocyte-macrophage phagocytosis assay. British Journal of Haematology. 1986; 62 :783-785 - 62.
Gallagher MT, Branch DR, Mison A, Petz LD. Evaluation of reticuloendothelial function in autoimmune hemolytic anemia using an in vitro assay of monocyte-macrophage interaction with erythrocytes. Experimental Hematology. 1983; 11 :82-89 - 63.
Noumsi GT, Billingsley KL, Moulds JM. Successful transfusion of antigen positive blood to alloimmunised patients using a monocyte monolayer assay. Transfusion Medicine. 2015; 25 :92-100 - 64.
Armstrong JK, Meiselman HJ, Fisher TC. Covalent binding of poly(ethylene glycol) (PEG) to the surface of red blood cells inhibits aggregation and reduces low shear blood viscosity. American Journal of Hematology. 1997; 56 :26-28 - 65.
Rampersad GC et al. Chemical compounds that target thiol-disulfide groups on mononuclear phagocytes inhibit immune mediated phagocytosis of red blood cells. Transfusion. 2005; 45 :384-393 - 66.
Karamatic Crew V et al. Two MER2-negative individuals with the same novel CD151 mutation and evidence for clinical significance of anti-MER2. Transfusion. 2008; 48 : 1912-1916 - 67.
Arndt PA, Garratty G. A retrospective analysis of the value of monocyte monolayer assay results for predicting the clinical significance of blood group alloantibodies. Transfusion. 2004; 44 :1273-1281 - 68.
Nance SJ, Arndt P, Garratty G. Predicting the clinical significance of red cell alloantibodies using a monocyte monolayer assay. Transfusion. 1987; 27 :449-452 - 69.
Bjorkholm M, Fagrell B, Przybelski R, Winslow N, Young M, Winslow RM. A phase I single blind clinical trial of a new oxygen transport agent (MP4), human hemoglobin modified with maleimide-activated polyethylene glycol. Haematologica. 2005; 90 :505-515 - 70.
Olofsson C et al. A multicenter clinical study of the safety and activity of maleimide-polyethylene glycol-modified hemoglobin (hemospan) in patients undergoing major orthopedic surgery. Anesthesiology. 2006; 105 :1153-1163 - 71.
Olofsson C et al. A randomized, single-blind, increasing dose safety trial of an oxygen-carrying plasma expander (hemospan) administered to orthopaedic surgery patients with spinal anaesthesia. Transfusion Medicine. 2008; 18 :28-39 - 72.
Vandegriff KD, Malavalli A, Mkrtchyan GM, Spann SN, Baker DA, Winslow RM. Sites of modification of hemospan, a poly(ethylene glycol)-modified human hemoglobin for use as an oxygen therapeutic. Bioconjugate Chemistry. 2008; 19 :2163-2170 - 73.
Vandegriff KD, Winslow RM. Hemospan: Design principles for a new class of oxygen therapeutic. Artificial Organs. 2009; 33 :133-138 - 74.
Young MA, Lohman J, Malavalli A, Vandegriff KD, Winslow RM. Hemospan improves outcome in a model of perioperative hemodilution and blood loss in the rat: Comparison with hydroxyethyl starch. Journal of Cardiothoracic and Vascular Anesthesia. 2009; 23 :339-347