E D I T O R I a L Editorial

E D I T O R I A L EDITORIAL

Universal RBCs

ntigens on RBCs have been a problem for blood RBCs; products such as Hb-based oxygen carriers are also transfusion right from the start. In the mid-17th universal, in that they can be transfused to any patient.) century, Jean Denis in France and Richard Lower Two general approaches could be used to make a uni- in England attempted animal-to-human blood versal RBC. The RBC antigens can be removed permanently transfusions,A only to be foiled by (among many factors) the from the RBC surface, or they can be masked to avoid rec- presence of incompatibilities between the animal RBCs and ognition by the immune system. This recognition by the human serum. James Blundell’s use of human-to-human immune system could lead to an immediate reaction via blood transfusions in the early 19th century lowered the im- preexisting antibodies in the recipient against antigens on munologic barrier, but major RBC antigenic differences be- donor RBCs or to later alloimmunization to these antigens. tween humans led to hemolytic reactions. With Landsteiner’s There has been substantial progress along both of these discovery of the ABO blood group antigens on RBCs and research directions, yielding RBC products of differing associated serum isoagglutinins a century ago,1 the major characteristics and different potential uses in transfusion cause of this immune reaction had been found, and suc- medicine. cessful blood transfusion could be achieved. Blood samples from donor and patient were each tested and classified into ENZYMATICALLY CONVERTED one of the four phenotypes—O, A, B, or AB—which allowed GROUP O CELLS the donor and patient phenotypes to be matched. At the turn of this new century, typing of ABO antigens still re- Work in the 1950s and 1960s by Morgan, Watkins, Kabat, and mains the critical step in RBC transfusion, necessitated by others elucidated the carbohydrate structures of the ABO anti- the presence of preexisting antibodies to A and/or B antigens on glycolipids and glycoproteins (reviewed by Hakomori2). gens in individuals lacking these antigens. The entire sys- It was shown that these antigens differed only in the termi- tem of blood collection, distribution, and transfusion re- nal monosaccharide attached to the precursor H oligosac- volves around the ABO blood groups. Elimination of this charide antigen: N-acetylgalactosamine for group A, galac- need for typing, by the creation of a universal RBC for trans- tose for group B, and no added monosaccharide for group fusion, would bring about a revolution in blood banking O (remains H antigen). This raised the possibility that A and akin to that which followed Landsteiner’s discovery of the B antigens could be converted back to H antigen by the re- ABO blood group system. So what are the potential meth- moval of the terminal monosaccharide unit by an appropri- ods with which to accomplish this task? Will universal RBCs ate exoglycosidase, α-N-acetylgalactosaminidase for group be coming to neighborhood blood centers and hospitals A and α-galactosidase for group B. Pioneering studies by the anytime soon? late Jack Goldstein and colleagues3 at the New York Blood The term “universal RBC” could have several mean- Center demonstrated that this process of enzymatic con- ings. Group O persons are considered universal RBC donors version of group B RBCs to group O could be carried out because they lack the antigens of the ABO blood group sys- with the enzyme coffee bean α-galactosidase under condi- tem, and thus their RBCs can be transfused to any recipi- tions that leave the RBCs physically and functionally intact and ent without concern for preexisting ABO antibodies. Blood suitable for transfusion. Studies in group O or group A vol- donor and recipient are also matched for D antigen, despite unteer recipients showed that these enzymatically converted the lack of anti-D in nonimmunized recipients, because the group O (ECO) cells functioned essentially identically to immunization rate is greater than 50 percent upon expo- group O RBCs; there was no evidence of acute transfusion sure of D– recipients to the D antigen. Thus, O– could be reactions or hemolysis, and RBC survival in the recipients considered the universal RBC. Finally, a small percentage was normal.4 Multi-unit or second transfusions were also of patients have preexisting antibodies to one or more of the well tolerated.5,6 An occasional serologic finding in the re- several hundred additional antigens described on RBCs, so cipients of these ECO cells was an increase in the titer of a truly universal RBC would have to be negative for all of anti-B or an incompatibility of recipient serum with ECO these antigens. (This discussion is limited to universal cells. As there was no evidence of associated clinical prob- lems, the meaning of these serologic results was not clear. TRANSFUSION 2000;40:1285-1289.

Volume 40, November 2000 TRANSFUSION 1285 EDITORIAL

In this issue of TRANSFUSION, Kruskall and col- matically converted to group O RBCs, which would increase leagues7 extend these studies by reporting a Phase II trial the blood supply, the ECO technology would not be eco- of the transfusion of ECO (B-to-O) RBCs to transfusion-de- nomically viable if used solely for this purpose. pendent patients of group O or A. In this crossover study of For ECO RBCs to become a universal blood supply, there 24 patients of group O or A, the patients were given either must be a process for A-to-O conversion. The original hope ECO or group O RBCs; 18 patients subsequently received a that this could be accomplished in a fashion parallel to the transfusion with the alternate product. Overall, the trans- B-to-O process by the use of an α-N-acetylgalactosamin- fusions of ECO RBCs appeared safe and effective; there were idase enzyme for A-to-O conversion has been dashed by the no acute transfusion reactions or evidence of hemolysis, biochemical complexity of the A antigen (see Fig. 1 in the and chromium survival studies showed that the lifespans article by Kruskall et al.7 in this issue of TRANSFUSION). of the transfused ECO and group O RBCs were equal. How- Epitopes of A can exist at both the terminal positions of the ever, a number of in vitro serologic abnormalities were oligosaccharide chains, where they can be removed by an noted, which were similar to observations in the previous α-N-acetylgalactosaminidase enzyme, and at internal po- studies of ECO RBC transfusions to normal volunteers.4,5 sitions, where they would be resistant to this exoglycosidase Five of 19 patients who received ECO RBCs had significant enzyme. Research aimed at the removal of all A epitopes is increases in anti-B titers, and 2 of these patients had incom- ongoing, but until this problem yields a biochemical solu- patible antiglobulin crossmatches with ECO RBCs.7 While tion, one must be guarded in predicting the future of a uni- the rising anti-B titers most certainly reflect an immune versal ECO blood supply. response to a small residual number of B epitopes on the Let us take a look at what future blood banking would ECO RBCs (compared to the original levels of approx. 0.8 × be like if the A-to-O conversion were accomplished and led 106/RBC), this weak antigen expression did not appear to to a totally group O blood supply, a mixture of ECO and cause measurable hemolysis in these patients. The incom- group O RBCs. It would be a completely different world, in patible crossmatches with ECO RBCs found with the serum which many of the daily activities in blood centers and from two patients is potentially concerning. Despite the hospitals would simply vanish. No more ABO to track for reassuring finding of normal transfusion results in these donors and patients. No more worries for recruiters at blood patients, additional work must be done to deduce the tar- centers about meeting separate targets for individual blood get of this antibody (residual B? neoantigen?) and its clini- groups, which can lead to overcollection to meet higher cal significance. Otherwise, the finding in this study7 that group O needs. No longer would inventory managers have 20 percent of group A patients and 40 percent of group O to ship blood around the country to redress the misalign- patients had incompatible crossmatches with ECO RBCs ment of blood group targets among regions. Nor would (often, weak reactions detectable only in the antiglobulin hospital transfusion services (or blood centers at each in- phase) would, at the least, introduce complicating serologic termediate transfer) have to reconfirm the ABO type of results into the testing protocol with an ECO blood supply, blood units. ABO transfusion errors, the most serious and and, more worrisome, might lead to reduced ECO RBC sur- life-threatening of transfusion reactions, would not occur. vival in vivo. These questions require further study. (But a caveat must be inserted here. Currently, ABO-incom- Nonetheless, it is fair to state that this overall research patible RBC transfusions occur because of error at some program has produced very strong and convincing data sup- stage of patient or blood unit identification, at an estimated porting the proposal that an ECO RBC produced by B-to-O rate of 1 in 33,000 transfusions, 6 percent of which have fatal conversion would serve as a universal RBC suitable for trans- outcomes.8 In a universal ECO blood supply, ABO identifi- fusion. Is this of any practical use to the blood supply? Sim- cation is no longer a problem, but the treatment process ply put, the answer is no. That is because the great benefit converting group A, B, or AB cells to group O is the concern. of ECO RBCs would be to convert the blood supply to a to- Rigorous attention to good manufacturing practices and tally group O supply. Shifting an extra 10 percent from group rigorous QC are critical to the avoidance of acute hemolytic B into the group O total and, potentially, another 5 percent transfusion reactions that could occur if the group A, B, or from group AB enzymatically converted to group A would AB antigens are not completely removed during the enzy- reduce the blood supply to an approximately equal mixture matic process to produce the ECO RBCs.) Overall, a univer- of group O and group A. This would do very little to allevi- sal ECO blood supply presents an appealing picture, but it ate the collection and inventory difficulties arising from the all must wait for the carbohydrate biochemists to solve the fact that blood donors have more than one ABO phenotype, A-to-O conversion problem. nor would it prevent the sometimes fatal complications of ABO transfusion errors. Moreover, no additional blood units STEALTH CELLS are actually produced, as the increased group O plus ECO (B-to-O) RBC supply must now support the larger group O While this research on ECO RBCs has proceeded for about plus group B patient population. Although the small percent- 25 years, other investigators9-12 over the past half-dozen age of group B units that normally outdate could be enzy- years have taken a different approach to universal RBCs by

1286 TRANSFUSION Volume 40, November 2000 EDITORIAL

attempting to mask the RBC antigens. This masking, or For a very new research field, this is quite a promising camouflaging, of antigens produces a “stealth” RBC that start. The key question is whether the PEG blockade can be cannot be detected by the immune system, which prevents increased without damage to the RBCs, and thereby a uni- antigen–antibody reactions with preexisting antibodies, as versal RBC can be created, or whether practical use can be well as alloimmunization to additional antigens.13-15 The made of a less than fully antigen-masked RBC in certain method is to treat the RBC surface with PEG, a neutral clinical transfusion circumstances. Promising new research polyether polymer with the chemical structure HO- is using a second-generation pegylation process, with

(CH2CH2O)n-CH2CH2OH. PEG exists in a variety of forms of changes in either the molecular weights and branching of differing molecular weights and branching patterns, and it the PEG13,17 or crosslinking of PEG with other proteins,18 to can be covalently coupled to proteins on the RBC surface produce PEG RBCs that have completely masked non-ABO through a number of linking compounds. The background antigens such as D, Kidd, and Duffy, as judged by antiglobu- for this work on RBCs was the demonstration that PEG lin testing and flow cytometry; exposure of ABO antigens is treatment of purified proteins reduced or eliminated their markedly reduced but clearly not eliminated. Again, this is immunogenicity, while leaving the proteins with normal tremendously exciting progress, but the PEG RBC field is function and showing no toxicity of these pegylated pro- much newer and less advanced than the ECO RBC field. teins when injected into animals or humans (indeed, a Hb- Collection of animal and, eventually, human data (compa- based oxygen carrier of potential benefit in transfusion rable to those from studies of ECO RBCs3-7) on the in vivo medicine is modified by PEG). PEG treatment of RBCs pro- results of PEG RBC transfusions and monitoring for trans- vides a protective shell around the RBC that excludes large fusion reactions, hemolysis, chromium survival studies, molecules, such as antibodies, but does not appear to in- antibody formation (including antibodies to neo-antigens hibit the interaction of the RBC with small molecules, such created by the pegylation process), and other potential tox- as glucose and oxygen, that are critical to RBC metabolism. icities will have to be performed to find out if the initial This PEG shell arises from extensive hydration coupled with promise of this research bears fruit. the flexibility and charge neutrality of PEG molecules; the If the research on PEG RBCs shows that they truly are net result is a large volume of exclusion around the RBC. stealth cells that evade the immune system, how can they Each of the four groups9-12 working on PEG RBCs took best be used in transfusion medicine? Stealth cells that somewhat different approaches in the exact chemistry completely mask the ABO antigens would be a rival univer- employed to produce PEG RBCs and in the in vitro and in sal RBC to ECO RBCs. Indeed, they would be a better prod- vivo animal testing that they used. Taken together, the set uct, for they would mask ABO and non-ABO antigens, but of initial results reported 3 or 4 years ago showed that PEG it is too early to predict whether this will be possible. Be- modification of the RBC surface could significantly reduce cause the results to date have shown complete masking of the antibody-mediated agglutination of PEG RBCs for both non-ABO antigens on PEG RBCs, as judged by in vitro tests, the ABO system and non-ABO systems including the Rh, it would be expected (but of course, must actually be shown Kell, Duffy, and Kidd systems. One group13 demonstrated in animal models and then in humans) that these PEG RBCs markedly reduced immunogenicity of PEG RBCs in a mouse would have essentially normal survival and markedly re- model, perhaps correlating with the reduced ability of ph- duced immunogenicity. There are clinical transfusion situ- agocytic cells to interact with the PEG RBCs. In vitro tests ations in which these RBCs would be a great benefit: in of the structure and function of the PEG RBCs suggested patients with antibodies to high-incidence antigens, a mix- that the RBCs (treated with low concentrations of PEG) had ture of antibodies to multiple antigens, or autoantibodies. not been significantly damaged by the pegylation and that PEG RBCs could be life-saving in a patient for whom no they retained their normal oxygen-binding and -transport compatible blood is available. In addition, the reduced or capacity9 and had normal RBC deformability.16 Limited in absent immunogenicity of the PEG RBC would be benefi- vivo studies of PEG RBCs in mice and rats showed that when cial in chronically transfused patients, such as those with RBCs were prepared with low concentrations of PEG, sur- sickle cell disease or thalassemia, who can have immuni- vival was normal, but treatment with higher concentrations zation rates of 30 percent or more. Transfusing these pa- of PEG resulted in poor in vivo survival.16 In addition, more tients with PEG RBCs would markedly reduce or eliminate sensitive in vitro tests of antigen recognition, including flow new immunizations. It is not clear if the blockade of D on cytometry and antiglobulin testing, clearly showed that the the stealth RBC will be sufficient for these RBCs to be truly antigens were still detectable on the PEG RBCs, especially considered D–; thus, stealth RBCs might not be suitable for for the ABO antigens.15 Nonetheless, these initial results transfusion to a 20-year-old woman who is D– and who, if were a clear proof that antigenicity and immunogenicity immunized against D, could have a future pregnancy in could be substantially reduced by PEG modification of the which HDN affects her baby. A potential additional benefit RBC surface. of PEG RBCs derives from the finding that these RBCs have

Volume 40, November 2000 TRANSFUSION 1287 EDITORIAL

reduced aggregation and a lower viscos- ity at low shear rates,12 so they could improve blood flow in vasoocclusive situations, such as arise in sickle cell disease.

INTO THE FUTURE As we assess this knowledge and peer into the future of this research, what do Fig. 1. The blood bank factory, circa 2005. A unit of RBCs prepared from the donor we see? ECO technology is further along (group A, D+ in this example) is processed by the blood bank factory, with successive and has undergone successful testing in machines performing WBC reduction, pathogen inactivation, enzymatic conversion humans, but the big challenge is to de- (removal) of the A antigen, and pegylation to mask all non-ABO antigens, including rive a biochemical process to achieve A- D. The final product, a WBC-reduced, pathogen-free, stealth ECO RBC, is a universal to-O conversion. Stealth technology has RBC for transfusion to any patient, regardless of ABO group, D phenotype, or the demonstrated that it can mask non-ABO presence of alloantibodies or autoantibodies to any RBC antigens. antigens as assessed in vitro, but so far it falls short on ABO antigens and has not yet been tried in any human transfusions. The results to REFERENCES date are exciting and hold tremendous promise. At this 01. Landsteiner K. Uber agglutinationserscheinungen time, a reasonable proposal would be to combine these normalen menslichen blutes. Wien Klin Wochenschr technologies, yielding stealth ECO RBCs. These could very 1901:14:1132-4. well be the true universal RBCs, with no (or very low) anti- 02. Hakomori S. Blood group ABH and Ii antigens of human genicity and immunogenicity for ABO and non-ABO anti- erythrocytes: chemistry, polymorphism, and their develop- gens and improved blood flow characteristics. mental change. Semin Hematol 1981;18:39-62. Universal RBCs would revolutionize our processes of 03. Goldstein J, Siviglia G, Hurst R, et al. Group B erythrocytes delivering blood from the donor to the patient. Many inven- enzymatically converted to group O survive normally in A, tory management issues that are associated with the B, and O individuals. Science 1982;215:168-70. present RBC donor supply consisting of multiple ABO phe- 04. Lenny LL, Hurst R, Goldstein J, et al. Single-unit transfu- notypes would vanish. RBCs would be readily available for sions of RBC enzymatically converted from group B to group O immunized patients who previously could not be provided to A and O normal volunteers. Blood 1991:77:1383-8. with crossmatch-compatible RBCs. This universal RBC sup- 05. Lenny LL, Hurst R, Goldstein J, Galbraith RA. Transfusions ply would be created by the blood bank factory of the fu- to group O subjects of 2 units of red cells enzymatically ture (Fig. 1). This factory, most likely located within or ad- converted from group B to group O. Transfusion jacent to the blood donor center, would take the donor 1994:34:209-14. RBCs and process them through a series of treatment steps. 06. Lenny LL, Hurst R, Zhu A, et al. Multiple-unit and second Inclusion of any technology in the blood bank factory will transfusions of red cells enzymatically converted from require clear evidence of significant clinical benefit and group B to group O: report on the end of Phase 1 trials. consideration of cost-benefit and risk-benefit issues. Al- Transfusion 1995;35:899-902. ready widespread is the process of prestorage WBC reduc- 07. Kruskall MS, AuBuchon JP, Anthony KY, et al. Transfusion tion (whether this should be a universal process is a sub- to blood group A and O patients of group B RBCs that have ject of controversy).19 Processes for pathogen inactivation been enzymatically converted to group O. Transfusion are currently undergoing clinical trials.20 The methods of 2000;40:1290-8. enzyme conversion and pegylation could produce the 08. Linden JV, Paul B, Dressler KP. A report of 104 transfusion stealth ECO RBC for universal RBC transfusion. The prom- errors in New York State. Transfusion 1992;32:601-6. ise of the future, the results of decades of basic and applied 09. Jeong ST, Byun SM. Decreased agglutinability of methoxy- research in transfusion medicine,21 the goal of improved polyethylene glycol attached red blood cells: significance as patient transfusion therapy—all could issue forth from this a blood substitute. Artif Cells Blood Substit Immobil blood bank factory. Biotechnol 1996;24:503-11. Douglas M. Lublin, MD, PhD 10. Hortin GL, Lok HT, Huang ST. Progress toward preparation Department of Pathology of universal donor red cells. Artif Cells Blood Substit and Immunology Immobil Biotechnol 1997;25:487-91. Washington University School of Medicine 11. Scott MD, Murad KL, Koumpouras F, et al. Chemical cam- St. Louis, MO 63110 ouflage of antigenic determinants: stealth erythrocytes. e-mail: [email protected] Proc Natl Acad Sci U S A 1997;94:7566-71.

1288 TRANSFUSION Volume 40, November 2000 EDITORIAL

12. Armstrong JK, Meiselman HJ, Fisher TC. Covalent binding 17. Fisher TC, Armstrong, JK, Meiselman JH, et al. Properties of of poly(ethylene glycol) (PEG) to the surface of red blood poly(ethylene glycol)-conjugated red blood cells. In: cells inhibits aggregation and reduces low shear blood vis- Tsuchida E, ed. Blood substitutes: present and future per- cosity. Am J Hematol 1997;56:26-8. spectives. Amsterdam: Elsevier Science, 1998:297-313. 13. Scott MD, Bradley AJ, Murad KL. Camouflaged blood cells: 18. Huang ST, Hortin GL, Huang Z. Coating of red blood cells low-technology bioengineering for transfusion medicine? with crosslinked polyethylene glycol (XPEG) inhibits agglu- Transfus Med Rev 2000;14:53-63. tination and shows favorable red cell survival (abstract). 14. Fisher TC. PEG-coated red blood cells—simplifying blood Transfusion 1998;38(Suppl):62S. transfusion in the new millenium? Immunohematology 19. Thurer RL, Luban NLC, AuBuchon JP, et al. Universal WBC 2000;16:37-58. reduction (letter). Transfusion 2000;40:751-2. 15. Garratty G. Stealth erythrocytes—a possible transfusion 20. Corash L. Inactivation of viruses, bacteria, protozoa, and product for the new century? Vox Sang 2000;78(Suppl leukocytes in platelet concentrates: current research per- 2):143-7. spectives. Transfus Med Rev 1999;13:18-30. 16. Murad KL, Mahany KL, Brugnara C, et al. Structural and 21. McCullough J. Research in transfusion medicine (editorial). functional consequences of antigenic modulation of red Transfusion 2000;40:1033-5. blood cells with methoxypoly(ethylene glycol). Blood 1999;93:2121-7.

Volume 40, November 2000 TRANSFUSION 1289