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EDITORIAL
EDITORIAL
Universal RBCs
A
ntigens on RBCs have been a problem for blood
transfusion right from the start. In the mid-17th
century, Jean Denis in France and Richard Lower
in England attempted animal-to-human blood
transfusions, only to be foiled by (among many factors) the
presence of incompatibilities between the animal RBCs and
human serum. James Blundell’s use of human-to-human
blood transfusions in the early 19th century lowered the immunologic barrier, but major RBC antigenic differences between humans led to hemolytic reactions. With Landsteiner’s
discovery of the ABO blood group antigens on RBCs and
associated serum isoagglutinins a century ago,1 the major
cause of this immune reaction had been found, and successful blood transfusion could be achieved. Blood samples
from donor and patient were each tested and classified into
one of the four phenotypes—O, A, B, or AB—which allowed
the donor and patient phenotypes to be matched. At the
turn of this new century, typing of ABO antigens still remains the critical step in RBC transfusion, necessitated by
the presence of preexisting antibodies to A and/or B antigens in individuals lacking these antigens. The entire system of blood collection, distribution, and transfusion revolves around the ABO blood groups. Elimination of this
need for typing, by the creation of a universal RBC for transfusion, would bring about a revolution in blood banking
akin to that which followed Landsteiner’s discovery of the
ABO blood group system. So what are the potential methods with which to accomplish this task? Will universal RBCs
be coming to neighborhood blood centers and hospitals
anytime soon?
The term “universal RBC” could have several meanings. Group O persons are considered universal RBC donors
because they lack the antigens of the ABO blood group system, and thus their RBCs can be transfused to any recipient without concern for preexisting ABO antibodies. Blood
donor and recipient are also matched for D antigen, despite
the lack of anti-D in nonimmunized recipients, because the
immunization rate is greater than 50 percent upon exposure of D– recipients to the D antigen. Thus, O– could be
considered the universal RBC. Finally, a small percentage
of patients have preexisting antibodies to one or more of the
several hundred additional antigens described on RBCs, so
a truly universal RBC would have to be negative for all of
these antigens. (This discussion is limited to universal
TRANSFUSION 2000;40:1285-1289.
RBCs; products such as Hb-based oxygen carriers are also
universal, in that they can be transfused to any patient.)
Two general approaches could be used to make a universal RBC. The RBC antigens can be removed permanently
from the RBC surface, or they can be masked to avoid recognition by the immune system. This recognition by the
immune system could lead to an immediate reaction via
preexisting antibodies in the recipient against antigens on
donor RBCs or to later alloimmunization to these antigens.
There has been substantial progress along both of these
research directions, yielding RBC products of differing
characteristics and different potential uses in transfusion
medicine.
ENZYMATICALLY CONVERTED
GROUP O CELLS
Work in the 1950s and 1960s by Morgan, Watkins, Kabat, and
others elucidated the carbohydrate structures of the ABO antigens on glycolipids and glycoproteins (reviewed by Hakomori2).
It was shown that these antigens differed only in the terminal monosaccharide attached to the precursor H oligosaccharide antigen: N-acetylgalactosamine for group A, galactose for group B, and no added monosaccharide for group
O (remains H antigen). This raised the possibility that A and
B antigens could be converted back to H antigen by the removal of the terminal monosaccharide unit by an appropriate exoglycosidase, α-N-acetylgalactosaminidase for group
A and α-galactosidase for group B. Pioneering studies by the
late Jack Goldstein and colleagues3 at the New York Blood
Center demonstrated that this process of enzymatic conversion of group B RBCs to group O could be carried out
with the enzyme coffee bean α-galactosidase under conditions that leave the RBCs physically and functionally intact and
suitable for transfusion. Studies in group O or group A volunteer recipients showed that these enzymatically converted
group O (ECO) cells functioned essentially identically to
group O RBCs; there was no evidence of acute transfusion
reactions or hemolysis, and RBC survival in the recipients
was normal.4 Multi-unit or second transfusions were also
well tolerated.5,6 An occasional serologic finding in the recipients of these ECO cells was an increase in the titer of
anti-B or an incompatibility of recipient serum with ECO
cells. As there was no evidence of associated clinical problems, the meaning of these serologic results was not clear.
Volume 40, November 2000 TRANSFUSION 1285
EDITORIAL
In this issue of TRANSFUSION, Kruskall and colleagues7 extend these studies by reporting a Phase II trial
of the transfusion of ECO (B-to-O) RBCs to transfusion-dependent patients of group O or A. In this crossover study of
24 patients of group O or A, the patients were given either
ECO or group O RBCs; 18 patients subsequently received a
transfusion with the alternate product. Overall, the transfusions of ECO RBCs appeared safe and effective; there were
no acute transfusion reactions or evidence of hemolysis,
and chromium survival studies showed that the lifespans
of the transfused ECO and group O RBCs were equal. However, a number of in vitro serologic abnormalities were
noted, which were similar to observations in the previous
studies of ECO RBC transfusions to normal volunteers.4,5
Five of 19 patients who received ECO RBCs had significant
increases in anti-B titers, and 2 of these patients had incompatible antiglobulin crossmatches with ECO RBCs.7 While
the rising anti-B titers most certainly reflect an immune
response to a small residual number of B epitopes on the
ECO RBCs (compared to the original levels of approx. 0.8 ×
106/RBC), this weak antigen expression did not appear to
cause measurable hemolysis in these patients. The incompatible crossmatches with ECO RBCs found with the serum
from two patients is potentially concerning. Despite the
reassuring finding of normal transfusion results in these
patients, additional work must be done to deduce the target of this antibody (residual B? neoantigen?) and its clinical significance. Otherwise, the finding in this study7 that
20 percent of group A patients and 40 percent of group O
patients had incompatible crossmatches with ECO RBCs
(often, weak reactions detectable only in the antiglobulin
phase) would, at the least, introduce complicating serologic
results into the testing protocol with an ECO blood supply,
and, more worrisome, might lead to reduced ECO RBC survival in vivo. These questions require further study.
Nonetheless, it is fair to state that this overall research
program has produced very strong and convincing data supporting the proposal that an ECO RBC produced by B-to-O
conversion would serve as a universal RBC suitable for transfusion. Is this of any practical use to the blood supply? Simply put, the answer is no. That is because the great benefit
of ECO RBCs would be to convert the blood supply to a totally group O supply. Shifting an extra 10 percent from group
B into the group O total and, potentially, another 5 percent
from group AB enzymatically converted to group A would
reduce the blood supply to an approximately equal mixture
of group O and group A. This would do very little to alleviate the collection and inventory difficulties arising from the
fact that blood donors have more than one ABO phenotype,
nor would it prevent the sometimes fatal complications of
ABO transfusion errors. Moreover, no additional blood units
are actually produced, as the increased group O plus ECO
(B-to-O) RBC supply must now support the larger group O
plus group B patient population. Although the small percentage of group B units that normally outdate could be enzy1286 TRANSFUSION Volume 40, November 2000
matically converted to group O RBCs, which would increase
the blood supply, the ECO technology would not be economically viable if used solely for this purpose.
For ECO RBCs to become a universal blood supply, there
must be a process for A-to-O conversion. The original hope
that this could be accomplished in a fashion parallel to the
B-to-O process by the use of an α-N-acetylgalactosaminidase enzyme for A-to-O conversion has been dashed by the
biochemical complexity of the A antigen (see Fig. 1 in the
article by Kruskall et al.7 in this issue of TRANSFUSION).
Epitopes of A can exist at both the terminal positions of the
oligosaccharide chains, where they can be removed by an
α-N-acetylgalactosaminidase enzyme, and at internal positions, where they would be resistant to this exoglycosidase
enzyme. Research aimed at the removal of all A epitopes is
ongoing, but until this problem yields a biochemical solution, one must be guarded in predicting the future of a universal ECO blood supply.
Let us take a look at what future blood banking would
be like if the A-to-O conversion were accomplished and led
to a totally group O blood supply, a mixture of ECO and
group O RBCs. It would be a completely different world, in
which many of the daily activities in blood centers and
hospitals would simply vanish. No more ABO to track for
donors and patients. No more worries for recruiters at blood
centers about meeting separate targets for individual blood
groups, which can lead to overcollection to meet higher
group O needs. No longer would inventory managers have
to ship blood around the country to redress the misalignment of blood group targets among regions. Nor would
hospital transfusion services (or blood centers at each intermediate transfer) have to reconfirm the ABO type of
blood units. ABO transfusion errors, the most serious and
life-threatening of transfusion reactions, would not occur.
(But a caveat must be inserted here. Currently, ABO-incompatible RBC transfusions occur because of error at some
stage of patient or blood unit identification, at an estimated
rate of 1 in 33,000 transfusions, 6 percent of which have fatal
outcomes.8 In a universal ECO blood supply, ABO identification is no longer a problem, but the treatment process
converting group A, B, or AB cells to group O is the concern.
Rigorous attention to good manufacturing practices and
rigorous QC are critical to the avoidance of acute hemolytic
transfusion reactions that could occur if the group A, B, or
AB antigens are not completely removed during the enzymatic process to produce the ECO RBCs.) Overall, a universal ECO blood supply presents an appealing picture, but it
all must wait for the carbohydrate biochemists to solve the
A-to-O conversion problem.
STEALTH CELLS
While this research on ECO RBCs has proceeded for about
25 years, other investigators9-12 over the past half-dozen
years have taken a different approach to universal RBCs by
EDITORIAL
attempting to mask the RBC antigens. This masking, or
camouflaging, of antigens produces a “stealth” RBC that
cannot be detected by the immune system, which prevents
antigen–antibody reactions with preexisting antibodies, as
well as alloimmunization to additional antigens.13-15 The
method is to treat the RBC surface with PEG, a neutral
polyether polymer with the chemical structure HO(CH2CH2O)n-CH2CH2OH. PEG exists in a variety of forms of
differing molecular weights and branching patterns, and it
can be covalently coupled to proteins on the RBC surface
through a number of linking compounds. The background
for this work on RBCs was the demonstration that PEG
treatment of purified proteins reduced or eliminated their
immunogenicity, while leaving the proteins with normal
function and showing no toxicity of these pegylated proteins when injected into animals or humans (indeed, a Hbbased oxygen carrier of potential benefit in transfusion
medicine is modified by PEG). PEG treatment of RBCs provides a protective shell around the RBC that excludes large
molecules, such as antibodies, but does not appear to inhibit the interaction of the RBC with small molecules, such
as glucose and oxygen, that are critical to RBC metabolism.
This PEG shell arises from extensive hydration coupled with
the flexibility and charge neutrality of PEG molecules; the
net result is a large volume of exclusion around the RBC.
Each of the four groups9-12 working on PEG RBCs took
somewhat different approaches in the exact chemistry
employed to produce PEG RBCs and in the in vitro and in
vivo animal testing that they used. Taken together, the set
of initial results reported 3 or 4 years ago showed that PEG
modification of the RBC surface could significantly reduce
the antibody-mediated agglutination of PEG RBCs for both
the ABO system and non-ABO systems including the Rh,
Kell, Duffy, and Kidd systems. One group13 demonstrated
markedly reduced immunogenicity of PEG RBCs in a mouse
model, perhaps correlating with the reduced ability of phagocytic cells to interact with the PEG RBCs. In vitro tests
of the structure and function of the PEG RBCs suggested
that the RBCs (treated with low concentrations of PEG) had
not been significantly damaged by the pegylation and that
they retained their normal oxygen-binding and -transport
capacity9 and had normal RBC deformability.16 Limited in
vivo studies of PEG RBCs in mice and rats showed that when
RBCs were prepared with low concentrations of PEG, survival was normal, but treatment with higher concentrations
of PEG resulted in poor in vivo survival.16 In addition, more
sensitive in vitro tests of antigen recognition, including flow
cytometry and antiglobulin testing, clearly showed that the
antigens were still detectable on the PEG RBCs, especially
for the ABO antigens.15 Nonetheless, these initial results
were a clear proof that antigenicity and immunogenicity
could be substantially reduced by PEG modification of the
RBC surface.
For a very new research field, this is quite a promising
start. The key question is whether the PEG blockade can be
increased without damage to the RBCs, and thereby a universal RBC can be created, or whether practical use can be
made of a less than fully antigen-masked RBC in certain
clinical transfusion circumstances. Promising new research
is using a second-generation pegylation process, with
changes in either the molecular weights and branching of
the PEG13,17 or crosslinking of PEG with other proteins,18 to
produce PEG RBCs that have completely masked non-ABO
antigens such as D, Kidd, and Duffy, as judged by antiglobulin testing and flow cytometry; exposure of ABO antigens is
markedly reduced but clearly not eliminated. Again, this is
tremendously exciting progress, but the PEG RBC field is
much newer and less advanced than the ECO RBC field.
Collection of animal and, eventually, human data (comparable to those from studies of ECO RBCs3-7) on the in vivo
results of PEG RBC transfusions and monitoring for transfusion reactions, hemolysis, chromium survival studies,
antibody formation (including antibodies to neo-antigens
created by the pegylation process), and other potential toxicities will have to be performed to find out if the initial
promise of this research bears fruit.
If the research on PEG RBCs shows that they truly are
stealth cells that evade the immune system, how can they
best be used in transfusion medicine? Stealth cells that
completely mask the ABO antigens would be a rival universal RBC to ECO RBCs. Indeed, they would be a better product, for they would mask ABO and non-ABO antigens, but
it is too early to predict whether this will be possible. Because the results to date have shown complete masking of
non-ABO antigens on PEG RBCs, as judged by in vitro tests,
it would be expected (but of course, must actually be shown
in animal models and then in humans) that these PEG RBCs
would have essentially normal survival and markedly reduced immunogenicity. There are clinical transfusion situations in which these RBCs would be a great benefit: in
patients with antibodies to high-incidence antigens, a mixture of antibodies to multiple antigens, or autoantibodies.
PEG RBCs could be life-saving in a patient for whom no
compatible blood is available. In addition, the reduced or
absent immunogenicity of the PEG RBC would be beneficial in chronically transfused patients, such as those with
sickle cell disease or thalassemia, who can have immunization rates of 30 percent or more. Transfusing these patients with PEG RBCs would markedly reduce or eliminate
new immunizations. It is not clear if the blockade of D on
the stealth RBC will be sufficient for these RBCs to be truly
considered D–; thus, stealth RBCs might not be suitable for
transfusion to a 20-year-old woman who is D– and who, if
immunized against D, could have a future pregnancy in
which HDN affects her baby. A potential additional benefit
of PEG RBCs derives from the finding that these RBCs have
Volume 40, November 2000 TRANSFUSION 1287
EDITORIAL
reduced aggregation and a lower viscosity 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 AD. 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
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Department of Pathology
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Washington University School of Medicine
St. Louis, MO 63110
e-mail: [email protected]
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