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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2121-2127
Structural and Functional Consequences of Antigenic Modulation of Red
Blood Cells With Methoxypoly(Ethylene Glycol)
By
Kari L. Murad,
Kathleen L. Mahany,
Carlo Brugnara,
Frans A. Kuypers,
John W. Eaton, and
Mark D. Scott
From Albany Medical College, Albany, NY; Children's Hospital,
Boston, MA; Children's Hospital Oakland Research Institute, Oakland,
CA; and Baylor College of Medicine, Houston, TX.
 |
ABSTRACT |
We previously showed that the covalent modification of the red blood
cell (RBC) surface with methoxypoly(ethylene glycol) [mPEG; MW ~5
kD] could significantly attenuate the immunologic recognition of
surface antigens. However, to make these antigenically silent RBC a
clinically viable option, the mPEG-modified RBC must maintain normal
cellular structure and functions. To this end, mPEG-derivatization was
found to have no significant detrimental effects on RBC structure or
function at concentrations that effectively blocked antigenic
recognition of a variety of RBC antigens. Importantly, RBC lysis,
morphology, and hemoglobin oxidation state were unaffected by
mPEG-modification. Furthermore, as shown by functional studies of Band
3, a major site of modification, PEG-binding does not affect protein
function, as evidenced by normal SO4 flux.
Similarly, Na+ and K+ homeostasis were
unaffected. The functional aspects of the mPEG-modified RBC were also
maintained, as evidenced by normal oxygen binding and cellular
deformability. Perhaps most importantly, mPEG-derivatized mouse RBC
showed normal in vivo survival (~50 days) with no sensitization after
repeated transfusions. These data further support the hypothesis that
the covalent attachment of nonimmunogenic materials (eg, mPEG) to
intact RBC may have significant application in transfusion medicine,
especially for the chronically transfused and/or allosensitized patient.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
BLOOD TRANSFUSIONS are a crucial
component of modern medical care, and, in general, most transfusions
occur without significant clinical consequences. An important exception
is in chronically transfused patients (eg, those with thalassemia and sickle cell disease) in whom allosensitization to minor (non-ABO/RhD) antigens occurs with high frequency (5% to 35%).1,2 In
particular, allosensitization is of special concern in the treatment of
sickle cell disease because of significant disparities in the
prevalence of a variety of non-ABO/RhD red blood cell (RBC) antigens
between the donor pool (typically white) and the affected patient
(typically of African descent). Indeed, some studies estimate that more
than 30% of sickle patients exhibit clinically significant
allosensitization (ie, severely shorted RBC survival
and/or mild-severe transfusion reactions) after repeated blood
transfusions to treat sickle crisis.1,2
The problem of allosensitization in the sickle population is likely to
increase as a consequence of new National Institutes of Health (NIH)
recommendations. Recent studies have clearly shown that a
regular regime of transfusion, such that less than 30% of the RBC are
sickle cell hemoglobin (HbS) positive, is an effective prophylactic
measure against stroke in at risk sickle patients.3 As a
result of these findings, an increased number of sickle patients will
likely develop clinically significant allosensitization. Consequently,
therapeutic interventions that diminish the risk of allosensitization
and/or allow for transfusions in an already allosensitized
patient will be crucial in formulating an effective transfusion therapy
for the sickle patient.
Previous work by our laboratory has shown that the covalent
modification of RBC surfaces with methoxypoly(ethylene glycol) [mPEG]
can effectively mask both ABO, RhD and non-ABO/RhD RBC (eg, C, c, E, S,
s) antigens and attenuate the antigenic recognition and immunogenicity
of the modified transfused cell.4-7 To determine if
mPEG-derivatization has any detrimental effects on RBC structure and
function, numerous RBC characteristics have been investigated. In
aggregate, the studies presented here show that derivatization of human
RBC at levels that effectively mask RBC antigens has little impact on
normal RBC structure and function.
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MATERIALS AND METHODS |
Blood collection.
After informed consent was obtained, whole blood was collected in
heparin, acid citrate dextrose (ACD), or EDTA from
laboratory volunteers and immediately processed. Volunteers were
selected to insure adequate representation, and no individuals were
excluded based on race or gender. Statistical analyses were performed
by the Student's t-test or analysis of variance
(ANOVA).8 Commercial anti-A and anti-B antisera were
obtained from Carolina Biological Supply (Burlington, NC). All other
biochemicals, unless otherwise noted, were obtained from Sigma Chemical
Co (St Louis, MO).
mPEG-derivatization.
Unless specifically stated, derivatization of erythrocytes was
performed as previously described.4,5 Briefly, washed RBC
were suspended to a hematocrit of approximately 12%
( 1.5 × 109 cells/mL) in isotonic alkaline phosphate
buffer (phosphate-buffered saline [PBS]: 50 mmol/L
K2HPO4 and 105 mmol/L NaCl) and incubated with
varying concentrations of cyanuric chloride-activated mPEG (MW 5 kD).
As indicated, the effects of pH (9.2 and 8.0), temperature (4°C and
25°C), and time (30 minutes and 60 minutes) on the efficacy of
derivatization were assessed. However, unless otherwise noted, all
functional and structural analyses used RBC that were reacted with mPEG
at pH 9.2, at 4°C for 30 minutes. Effects of storage on the efficacy
of RBC derivatization were assessed by storing blood at 4°C in ACD
vacutainer tubes for up to 45 days.
Effects of mPEG-derivatization on antigenic recognition.
Antigenic camouflage was assessed by attenuation of anti-A and anti-B
human antisera-mediated RBC aggregation using a platelet aggregometer
(Chrono-Log, Havertown, PA), as previously described.4,5 Briefly, 450 µL of an RBC suspension (6% hematocrit in isotonic saline) was placed in an aggregometer cuvette at 37°C, with stirring, and 20 µL of anti-A and/or anti-B typing serum (or
autologous/heterologous serum) was added. RBC aggregation was then
observed over time. Our previous studies show a direct relationship
between effects on aggregation and measured binding of appropriate
antisera to intact RBC.4-7
Structural studies.
To test the integrity of mPEG-derivatized RBC, changes in gross
morphology, membrane stability, deformability, ion exchange, and RBC
membrane proteins were examined. RBC indices of control and
mPEG-modified RBC were determined using a Swelab 920EO hematology analyzer (DiaPharma, Franklin, OH). The morphology of both control and
mPEG-derivatized cells was assessed by both light and scanning electron
microscopy (SEM), as previously described.4 Spontaneous lysis was measured by determining total hemoglobin concentration spectrophotometrically (540 nm) for both the total cell suspension and
the cell supernatant using Drabkin's reagent.9
Gross changes in the membrane protein pattern were analyzed by
one-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) of membrane ghosts, as previously
described.4,10-12 Functional integrity of the RBC membrane,
as well as functioning of membrane pumps, was assessed by ion
homeostasis. Cation (Na+, K+) and anion
(SO42) fluxes were examined by flame
photometry in accordance with standard procedures.13,14
Cellular deformability was determined using a Technicon ektacytometer
(Bayer Diagnostics, Tarrytown, NY). Ektacytometry is a sensitive method
for detecting population changes in deformability based on changes in
cell geometry, surface area, cytoplasmic viscosity, and cellular
hydration.15-17 In brief, control and mPEG-derivatized RBC
were suspended in 4% polyvinylpyrrolidone. A constant shear stress of
125 dyne/cm2 was applied to the cells, and the osmolality
of the suspending medium was gradually changed over a range of 100 to
500 mOsm/kg.
In vivo survival studies.
In vivo studies provide the ultimate indication of the normality (ie,
viable structure and function) of PEG-derivatized RBC. Consequently,
mice (Balb/c) were transfused as previously described.4,18 The concentrations of mPEG used to treat murine RBC in these
experiments ranged from 0 to 5 mmol/L. Murine RBC were labeled using a
fluorescent, membrane-anchoring marker, PKH-26 (Sigma Chemical Co).
Equal numbers of control and mPEG-derivatized RBC (40% hematocrit)
were injected intraperitoneally (IP; a common mode of transfusion in
veterinary medicine) into recipient Balb/c mice. Blood samples from the
recipient mice were observed until the labeled RBC were cleared from
circulation (approximately 40 to 50 days for allotransfusions).
Survival of fluorescently labeled mPEG-treated and control RBC was
monitored by analyzing the percentage of fluorescently labeled RBC by
flow cytometry.18 In some studies, mice were
"hypertransfused" using the above protocol to determine the
effects of a high hematocrit of mPEG-modified RBC. Because
hypertransfusion results in suppression of endogenous RBC production,
this procedure allowed for an almost complete blood exchange. Mice were
hypertransfused by injecting (IP) RBC at 3- to 5-day intervals for 62 days. The percentage of PKH-26-labeled RBC in mice receiving either
control or mPEG-modified (0.4 mmol/L) RBC was determined by flow
cytometry at 24 hours posttransfusion.
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RESULTS |
As we have previously shown, mPEG-derivatization decreased
antisera-induced aggregation in a dose-dependent manner.4-7
These previous studies derivatized the RBC at a pH of 9.2 for 30 minutes at 4°C. This high pH was used because it was previously
reported to be the optimal pH of the chemical reaction between cyanuric chloride-activated mPEG.19 To determine whether human RBC
could be efficiently modified at a more physiological pH and under less restrictive conditions, we examined the effects of pH, derivatization time, and temperature on antisera-mediated aggregation. As shown in Fig
1A, the derivatization process is quite
malleable. Indeed, derivatization at pH 8.0 for 60 minutes was found to
consistently result in a more efficient antigen masking, as assessed by
antisera-mediated aggregation. In addition, as shown in Fig 1B,
mPEG-modified RBC effectively inhibit antisera-mediated aggregation of
unmodified cells in a dose-dependent manner.
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| Fig 1.
(A) Changes in pH, temperature, and derivatization time
did not effect the ability of human RBC to be effectively modified with
mPEG (1.2 mmol/L), as shown by comparable inhibition of
antiserum-mediated aggregation. (B) Furthermore, addition of
mPEG-modified (2.4 mmol/L) RBC to control cells diminished
antisera-mediated aggregation in a dose-dependent manner. The values
shown are the percentage of mPEG-modified RBC. The above figures are
representative of more than 5 independent assays.
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Stored RBC were found to be readily derivatized even after storage at
4°C for 0, 7, and 45 days (Fig 2).
Importantly, the derivatization process had no significant effects on
RBC lysis (<0.5% lysis even in the presence of 10 mmol/L activated
mPEG) or hemoglobin oxidation (>98% oxyhemoglobin after
derivatization). Indeed, morphologically, the derivatized RBC appeared
normal by both light microscopy and SEM (Fig
3). Analysis of RBC indices also showed no
overall alterations arising from mPEG-modification (Table
1). Although the mPEG-modified cells showed
a tendency towards slightly (but not significantly) decreased
intracellular K+ concentrations relative to the pH control,
only at the highest levels of derivatization (>5 mmol/L mPEG) did one
see a significant increase in intracellular Na+ (Table 1).
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| Fig 2.
RBC were readily modifiable by activated mPEG after
extended periods of storage. However, a slight decrease in the
inhibition of antisera-mediated aggregation was observed over the 45 days of storage. Shown are the aggregation curves of RBC following 0 (A), 7 (B), and 45 days (C) of storage in ACD at 4°C. RBC were
derivatized with 0 to 1.2 mmol/L cyanuric chloride-activated mPEG at
4°C for 30 minutes in PBS (pH 9.2).
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| Fig 3.
Normal morphology was observed in the mPEG-RBC. Shown are
scanning electron micrographs (×2,300) of control and
mPEG-derivatized (1.2 mmol/L) RBC. RBC were derivatized with 1.2 mmol/L
cyanuric chloride-activated mPEG at 4°C for 30 minutes in PBS (pH
9.2).
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The above findings are consistent with our earlier reports indicating
normal lysis rates and osmotic fragility after in vitro incubations of
up to 48 hours.4,5 Similarly, oxygen binding was unaffected
by mPEG-derivatization of the membrane. In agreement with recently
published findings from our Korean colleagues,20 the
P50 of the mPEG-modified RBC was unchanged from that of the control cells previously subjected to pH 9.2 conditions
(P50 = 22.3 and 23.0, respectively).
Closely related to cell shape and membrane stability is cation and
anion homeostasis. Ion homeostasis is governed in large part by
integral membrane proteins that are subject to mPEG-derivatization. As
we previously showed, the anion transporter Band 3 is a major site of
mPEG-binding.4 Consequently, to determine if derivatization adversely affected Band 3 function,
35SO4 influx in the absence and
presence of the Band 3 inhibitor di-isothiocyano-disulfonyl stilbene
(DIDS) was examined. As shown in Fig 4A,
RBC derivatization with up to 5 mmol/L mPEG did not alter
35SO4 influx. Furthermore, Band
3 dependent anion transport even in the pegylated cellswas readily
inhibited by DIDS.
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| Fig 4.
Anion (A) and cation (B) permeability and transport were
unaffected by mPEG-modification. (A) As previously reported, Band 3 is
a common site of derivatization with cyanuric chloride-activated mPEG.
However, Band 3 function was observed to be unimpaired after
derivatization, as shown by normal 35SO4 influx
( ). In addition, no difference was observed in the ability of the
DIDS to inhibit the transporter function of Band 3 ( ). (B)
Similarly, the RBC Na+-K+ pump was
unaffected by mPEG-derivatization. Shown are control () and ouabain
(0.1 mmol/L; )-treated RBC. As with DIDS, ouabain was also found to
have no differential effect on the control and mPEG-modified cells. The
finding that no significant difference was observed in the sensitivity
of either pump to small inhibitors (DIDS and ouabain) shows the normal
flux of small, but not large, molecules across the RBC surface. The
results shown are the average ± standard deviation of three separate
experiments.
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Similarly, active cation transport was unaffected by
mPEG-derivatization, as shown by the ouabain-sensitive Na+
efflux, which measures the maximal functional capacity of the Na+-K+ adenosine triphosphatase (ATPase) (Fig
4B). There were also no changes induced by mPEG-derivatization on the
maximal activity of the
Na+-K+-Cl cotransport system
(expressed as bumetanide-sensitive Na+ or K+
effluxes, data not shown). No effects of mPEG-derivatization could be
shown on the passive permeability of the erythrocyte. In particular,
the efflux of K+ from fresh cells in the presence of 0.1 mmol/L ouabain and 0.01 mmol/L bumetanide was unaffected by
mPEG-derivatization (data not shown). The finding that transport
inhibitors like DIDS, ouabain, and bumetanide could readily inhibit
their targets further attests to the functional integrity of integral
membrane proteins of pegylated RBCs. Furthermore, the results obtained
with these inhibitors suggest that other small molecules (eg, oxygen,
glucose) can access and interact with the membrane of pegylated RBC,
whereas large molecules (eg, IgG) and cells cannot.
Functionally related to ion homeostasis (via cell size and
intracellular viscosity) and membrane integrity is the ability of RBC
to deform. The ability of the erythrocyte to undergo deformation is
crucial to not only its physiological function of delivering oxygen,
but also to its survival within the circulatory system. Indeed, genetic
conditions (eg, sickle hemoglobin) or pharmacological agents (eg,
membrane oxidants) that affect cellular deformability have been shown
to have profound effects on erythrocyte function and
survival.16,17,21 Hence, because the membrane of the
mPEG-treated cell undergoes significant surface modification, we
examined what, if any, effects variable levels of mPEG modification
would have on RBC deformability using ektacytometry. As shown in Fig
5, only at very high levels of
derivatization ( 5 mmol/L mPEG) did the deformability profile of the
modified cells show any significant differences from the control RBC.
Of particular interest in analyzing the ektacytometric curve is the
maximum deformability index (DImax) at isotonicity
(~290 mOsm). As shown, all samples with the exception of the 5 mmol/L-modified sample fall within the normal range (0.45 ± 0.08).
However, even in the 5 mmol/L-treated samples, the deformability is
still significantly better than that seen in sickle,  thalassemic, or slightly oxidant stressed glucose 6 phosphate dehydrogenase (G6PD)-deficient RBC.16,21 All other parameters
(0min = Minimum DI and is where 50% hemolysis is
observed in a classical osmotic fragility test;
0hyp = 1/2DImax and arises from the
hypertonic osmolality and resultant cell shrinkage) are within the
normal range.
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| Fig 5.
mPEG derivatization of human RBC had no significant
effect on RBC deformability at dosages (0.6 mmol/L to 2.4 mmol/L),
which effectively block/inhibit antigenic recognition and
immunogenicity. Ektacytometry was performed as previously
reported.16,17 All samples were stored for 24 hours at
4°C before analysis.
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In aggregate, all in vitro studies suggested that the mPEG-modified RBC
show normal structure and function. However, the ultimate test as to
the normality of modified RBC is in vivo survival. To examine this
question, we used a murine (Balb/c) model using the transfusion of
syngeneic control and mPEG-modified cells. With this model, we examined
the effects of the degree of mPEG-derivatization and whether
mPEG-modified cells could efficaciously replace normal, unmodified RBC.
Despite the inherent fragility of mouse erythrocytes, a significant
degree of derivatization was possible; though as shown in Fig
6, it was possible to overmodify the RBC,
such that a more rapid clearance of the transfused RBC occurred. How
this finding relates to human RBC is somewhat unclear at this point. However, it is important to note that significant in vitro lysis occurred in the murine cells at mPEG concentrations 0.6 mmol/L. In
contrast, under the same conditions, significant lysis of human RBC
only occurred at mPEG concentrations 12 mmol/L. Furthermore, as we
previously reported, repeated transfusion of mice with mPEG-modified (0.4 mmol/L) RBC did not sensitize the animals to the modified RBC, and
these cells showed normal survival even after five transfusions each
separated by a minimum of 50 days.
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| Fig 6.
The in vivo survival of mPEG-modified murine RBC was
unaffected by derivatization at low concentrations of mPEG. Shown are
the clearance rates of primary IP infusions of control and
mPEG-modified (0.2 to 0.8 mmol/L) RBC. Survival was observed using a
fluorescent fatty acid label (PKH-26), as previously
described.4,5 Each transfusion approximated 8% to 10% of
total mouse RBC mass. The results shown are the mean ± standard
deviation of six Balb/c mice per mPEG condition, and the clearance
slopes were determined by linear regression. (INSERT) Hypertransfusion
of mice with autologous mPEG-modified RBC had no effect on mouse
viability. Multiple transfusions of Balb/c mice (n = 6 per group)
with mPEG-modified (0.4 mmol/L) or control RBC resulted in no apparent
differences between groups in terms of viability or behavioral and
physical activity. These data suggest a lack of toxicity and emphasize
the in vivo normality of mPEG-modified RBC. The mice received a total
of 33 transfusions (400 µL each of a 40% hematocrit) of control or
mPEG-modified RBC.
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Additional studies further showed that mPEG-derivatized murine RBC, at
levels that yielded normal in vivo RBC survival (shown is 0.4 mmol/L
mPEG), were readily and safely tolerated in hypertransfused mice (Fig 6
insert). In this study, the total time from the initial transfusion to
the final clearance of the transfused RBC was approximately 140 days,
with a maximum of approximately 80% of the mouse RBC volume consisting
of mPEG or labeled control RBC. During this time, the PEG-transfused
mice received a total of 6.7 g of mPEG per kg body weight and showed no
evident toxicity relative to the mice hypertransfused with control RBC.
However, both populations of mice showed secondary iron overload to the
repeated transfusions. On necropsy, gross examination of the organs
(with one exception discussed below) showed no significant differences
in appearance between the three groups (nontransfused,
control-transfused, mPEG-RBC transfused). Furthermore, no differences
in body weight or the wet weight of the liver, kidney, heart, or brain
were noted between the mice hypertransfused with control cells (Table
2). Interestingly, however, the mice
hypertransfused with control-RBC showed a significant increase in
spleen size, whereas the mPEG-transfused animals showed no increase in
the spleen relative to the nontransfused animals.
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DISCUSSION |
Despite medical advances in transfusion medicine, transfusion reactions
are still a clinically significant problem. Although the NIH estimates
that 1:100,000 transfusions result in a fatal rejection
reaction,22 it is estimated that 1 of every 4,000 units
transfused results in a nonfatal transfusion reaction (eg, accelerated
clearance of transfused RBC).23 The majority of these
clinically significant events arise because of the presence of
antibodies to non-ABO/RhD RBC antigens that are not typically screened
for by blood banks. Even in those patients at greatest risk of
allosensitization, ie, the chronically transfused, debate continues as
to the efficacy of pretransfusion matching of donor blood and
prevention of clinically significant sequela, as well as subsequent
allosensitization. In part, this debate arises from the expense of
laboratory testing and the sheer abundance (>300) of potentially
immunogenic non-ABO/RhD blood group antigens.24,25 Consequently, a need continues to exist to diminish adverse immune reactions while providing the necessary oxygen carrying capacity in the
chronically transfused patient.
Because transfusion rejections are immune-mediated responses arising
from the presence of antigenic proteins, glycoproteins, and glycolipids
located on the RBC membrane, significant research over the last 20 years has been devoted to the development of acellular blood
substitutes lacking these surface antigens.26,27 Though
commendable in theory, the safety and efficacy of
acellular/liposome-encapsulated blood substitutes is still in
question.28 Indeed, the short circulating life as well as
secondary iron toxicity after repeated transfusions with these agents
is of major concern. In contrast, our work on PEG-modified RBC is aimed
at maintaining cellular integrity, longevity, and the myriad of other
functions of the RBC while reducing its inherent antigenicity and
immunogenicity by physically masking membrane antigenic sites.
Hence, establishing a balance between decreased cellular antigenicity
and normal RBC structure and function is a major concern in
PEG-modification of cells. Indeed, the need for balance is shown by the
fact that at high levels of derivatization, human and murine RBC show
some loss of function. Importantly though, these adverse structural and
functional effects in human RBC occur at derivatization dosages (>5
mmol/L) well in excess of that needed to effectively attenuate
antigenic recognition of RBC antigens as shown in Figs 3 through 5.
Indeed, the ability to globally camouflage the RBC surface should be
particularly desirable in transfusion medicine because of the sheer
abundance of RBC antigens, their ability to mediate a potent immune
response, and the difficulty/expense of adequately screening donors and
recipients. Effective PEG coverage of the cell surface also diminishes
cell-cell interactions. In mixed cell populations (Fig 1B),
mPEG-modified cells significantly diminished aggregation of the
unmodified RBC in response to antisera. Others have shown that
PEG-derivatization of human RBC greatly reduces the low shear viscosity
of these cells when suspended in autologous plasma.29 These
findings may be of particular interest in the treatment of diseases
such as sickle cell anemia, in which vasocclusive eventsmediated by
vascular wall adherence and RBC aggregate formationplay an important
role in the disease pathology. Use of the less "adhesive" PEG-RBC
may make it possible to transfuse sickle patients at a lower level than
the new NIH guidelines recommend because of the antiaggregation effect
of PEG-RBC.
Of potential concern is whether the chronic administration of
mPEG-modified RBC is safe. As shown in this study, mice hypertransfused with mPEG-RBC showed no obvious toxicity. Furthermore, based on projections of human dosages, annual PEG administration (eg, to chronically transfused adult sickle or thalassemic patients) will range
from 230 to 760 g of PEG (based on 18 and 30 U of blood per year,
respectively). Using a mean human body weight of 68.2 kg (150 lb), this
results in an annual PEG dosage of 3.4 to 11.1 g/kg. This is
substantially less than the amount of PEG administered to the
hypertransfused mice when adjusted to an annual rate (ie, 28 g/kg body
weight). Hence, the lack of observed toxicity in our repetitively
transfused mice suggestsbut does not provethat the acute and
short-term transfusion of PEG-RBC should be safe and well tolerated in
humans. Unresolved is the question as to whether the long-term (eg, 10 years) administration of PEG-RBC to the chronically transfused patient
will be safe. However, in support of its potential safety, animal
models have shown that PEG (up to approximately 10 kD) and
PEG-conjugated proteins (eg, PEG-Hb) are effectively excreted via the
kidneys, and thus, would not likely accumulate within the transfused
patient.30
Finally, as shown in Fig 6, extensive derivatization of murine cells
did result in foreshortened in vivo survival. While human, canine, and
sheep RBC show much greater in vitro "tolerance" to mPEG-modification than the inherently fragile murine cell, it may be
that to effectively camouflage all RBC antigens (eg, the A and B
determinants to yield a "universal" RBC), some decrement in
circulation time might be observed. This, however, might be tolerable
because in profoundly anemic patients already allosensitized, transfusion of lysis-resistant RBC that effectively oxygenate the
tissues would still be of significant benefit. Furthermore, if these
cells prove efficacious in diminishing the risk of allosensitization, a
moderate decrease in the in vivo survival would also be acceptable, because the consequences of sensitization are so much greater.
In summary, our immediate goal in developing PEG-derivatized RBC is not
to necessarily create a universal RBC, but rather to produce
antigenically and immunogenically attenuated cells with normal or near
normal in vivo survival for use in the chronically transfused or
allosensitized patient. To this end, we have shown that mPEG
derivatization has no detrimental effect on RBC structure or function
(morphology, membrane stability, P50, ion homeostasis, and
cellular deformability) at concentrations that significantly reduce RBC
antigenicity and immunogenicity.4-7 Thus, use of these "stealth erythrocytes"4 in the chronically transfused
or in patients with rare blood types may be effective at both
delivering oxygen as well as attenuating the risk of allosensitization.
| |
ACKNOWLEDGMENT |
The authors thank Simone Petrocine, Todd P. Christian, Dave L. Devernoe, Scott G. Menzie, and Stanley P. Mudzinski for technical assistance and advice.
| |
FOOTNOTES |
Submitted July 29, 1998; accepted November 12, 1998.
Supported by National Institutes of Health grants: HL53066 (M.D.S.),
HL58584 (M.D.S.), HL55213 (F.A.K.), and HL32094 (F.A.K.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mark D. Scott, PhD, Division of
Experimental Pathology, Department of Pathology and Laboratory Medicine
MC-81, Albany Medical College, 47 New Scotland Ave, Albany, NY 12208.
| |
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ABSTRACT
INTRODUCTION