complement leads to a process that he termed the “acti-
vation” of endothelium. The endothelium is rendered
permeable, allowing penetration of immunocompetent
cells, leading to the characteristic picture of inflamma-
tion, thrombosis and ischemia . AHR is less fre-
quently encountered than acute cellular rejection (ACR).
The latter responds well to therapy directed against T
lymphocytes as a preventive and therapeutic strategy .
AHR is relatively unresponsive to therapies that target T
lymphocytes. Treatment focuses rather on removal of
preformed alloantibodies against donor-specific human
leukocyte antigens (HLA). This can be accomplished by
means of plasmapheresis (PP) in combination with im-
munosuppressive agents that inhibit B-cell proliferation,
such as mycophenolate.
In renal transplantation, AHR has a poor prognosis for
immediate graft survival . Grafts that do survive are
subject to impaired long-term allograft function, and
patients experience early allograft loss [3, 7]. Reports
that used conventional therapy reveal that 1-year graft
survival does not exceed 15%–50% [8, 9]. New treat-
ment strategies that use intravenous immunoglobulin
(IVIG) have been found to be more efficacious. Several
studies, including a recently published report by our
group [10–13], describe the combined use of PP or other
means of immunoadsorption in conjunction with IVIG
and standard maintenance immunosuppression.
IVIG has immunomodulatory properties and is effec-
tive in treating several autoimmune and inflammatory
conditions such as immune thrombocytopenic purpura,
hemolytic anemias, and autoimmune neutropenias .
Various actions of IVIG have specific relevance to alloan-
tibody-mediated acute rejection of transplanted allo-
grafts. These include neutralization of autoantibodies
, inhibition of activation of endothelial cells ,
downregulation of antibody synthesis as a result of inhi-
bition of B- and T-cell proliferation [17–19], and in-
creased apoptosis of B cells . These properties of
IVIG may explain its role as a helpful adjunct in the
treatment of AHR.
In our experience with AHR in renal allografts, the
combined use of IVIG and PP is associated with a 1-year
graft survival of 81% . In the current study we
describe the extension of that experience by expanding
the study group and follow-up. We now describe 23
patients with AHR who were treated with PP and IVIG,
and we report outcome data extending more than 2
MATERIALS AND METHODS
Our study group consisted of all consecutive kidney or
kidney-pancreas transplants performed at Duke Univer-
sity Medical Center between January 1999 and August
2003 (n ϭ 519). Data collection consisted of a review of
medical records. We had a preexisting database contain-
ing demographic and clinical information on all patients
transplanted between January 1999 and August 2001;
data for patients transplanted between August 2001 and
August 2003 were added to this database. Follow-up for
all patients was extended to August 2004.
Pathology and Immunopathology
Biopsies were performed to evaluate allograft dysfunc-
tion; protocol biopsies were not performed. Biopsy re-
sults were used to classify patients into rejection groups,
namely ACR or AHR. Patients with no biopsy-proven
evidence of rejection or who did not undergo biopsy were
assigned to the no rejection group (No REJ). Transplant
biopsy samples were routinely processed and stained by
hematoxylin-eosin, periodic acid–Schiff, methenamine
silver, and Masson trichrome methods. ACR was diag-
nosed and graded according to the Banff 1997 criteria
. The diagnosis of AHR was suggested by the fol-
lowing histologic criteria: interstitial infiltrate of inflam-
matory cells (1) involving more than 25% of biopsy
tissue, (2) with predominant focus on peritubular capil-
laries, (3) composed at least in part of neutrophils, and
(4) associated with minimal tubulitis. AHR was con-
firmed on the basis of criteria recently published by the
Banff group . Patients who had biopsy-proven typ-
ical histology for AHR and who had either positive
staining for C4d or presence of donor-specific HLA an-
tibodies were considered to have confirmed AHR. C4d
staining was performed on a frozen section by means of
a mouse monoclonal anti-C4d antibody (Biogenesis, San-
down, NH). Renal transplant biopsy samples after Feb-
ruary 2001 were routinely stained with anti-C4d anti-
body. Stored frozen tissue from biopsies performed
between January 1999 and February 2001 were retro-
spectively stained for C4d.
At the time of initial workup, sera from all kidney
transplant candidates were evaluated for the presence of
anti-HLA immunoglobulin (Ig) G antibodies by means
of both the complement-dependent cytotoxicity tech-
nique enhanced with antihuman globulin (CDC-AHG)
and flow cytometry techniques (flow panel-reactive an-
tibody [PRA]) . The CDC-AHG technique for T-
cell panel analysis and the complement dependent cyto-
toxicity (CDC) (NIH modified, three wash) technique for
B-cell panel analysis were used to determine whether any
antibody detected had cytotoxic properties or whether
any IgM was present. Dithiothreitol was used to reduce
any IgM present, thereby allowing for the detection of
IgG antibodies by the cytotoxic techniques. Because flow
cytometry is three times more sensitive than the CDC-
AHG technique, it was used to determine the presence of
351IVIG and Plasmapheresis in Humoral Rejection
any HLA-directed antibody. Also, the flow cytometry
technique can specifically identify HLA class I– versus
class II–directed IgG antibodies.
The fine specificity of any antibody detected was
analyzed by flow cytometry by means of specificity
beads, latex beads coated with class I or class II HLA
molecules, as well as single antigen beads and latex beads
coated with molecules of a single HLA class I or class II
allele (One Lambda, Canoga Park, CA). In this manner,
the exact specificity of the antibody can be determined to
identify alleles in the donor population that would be
The final cross match included a CDC-AHG T-cell
and CDC (three wash) B-cell cross match for all donor-
recipient combinations, and a flow cytometry T- and
B-cell cross match for all recipients with HLA-directed
antibodies detected by flow cytometry.
PRAs before transplantation were obtained on all
patients. CDC-AHG, enzyme-linked immunosorbent as-
say, and flow cytometry techniques were used, and peak
historic PRAs were recorded for the purpose of this
study. If flow cytometry PRAs were available (they were
routinely used starting in January 2000), they were
reported. If flow cytometry PRAs were not available,
enzyme-linked immunosorbent assay or CDC-AHG
PRAs were reported. Flow cytometry PRAs were avail-
able in the majority of patients. For simplicity of pre-
sentation, the results are presented as T- and B-cell
PRAs, regardless of the method used.
Patient Characteristics, Treatment, and Outcome
Charts were screened for demographic patient character-
istics, namely sex, race, age, and type of transplant (liv-
ing donor or deceased donor). Furthermore, the following
transplant-specific data were extracted: cold ischemia
time, presence of delayed graft function (defined as the
need for renal replacement therapy in the first week after
transplantation), type of induction therapy (daclizumab
or antithymocyte globulin), historic peak T- and B-cell
PRA, and modality of treatment (PP and IVIG, PP
alone, IVIG alone, pulse methylprednisolone, antithy-
mocyte globulin, and muromonab-CD3).
Maintenance immunosuppression in all patients con-
sisted of a calcineurin inhibitor (tacrolimus or cyclospor-
ine), mycophenolate, and prednisone. Patients who were
identified as having AHR received a combined regimen
of PP and IVIG (Gamimune, Bayer Biological Products,
Research Triangle Park, NC; or Venoglobulin, Alpha
Therapeutic, Los Angeles, CA). A typical PP regimen
consisted of four daily sessions (range 3–6 days) with 5%
human albumin replacement. The number of PP sessions
was based on the clinical response to therapy as measured
by urine output and serum creatinine. IVIG, usually at a
dose of 2 g/kg, was administered after the last PP session.
However, there was a wide dose variation.
The primary outcome measure was return to renal
replacement therapy after kidney transplantation. Sec-
ondary outcome measures were last serum creatinine at
end of follow-up and patient survival.
The results were summarized as mean Ϯ SEM or median
and interquartile range. Continuous variables were com-
pared by the two-tailed unpaired t-test, and dichotomous
variables were compared using 2 ϫ 2 contingency tables
and Fischer’s exact test. Survival analysis was performed
with the Kaplan-Meier method, and comparisons be-
tween survival curves were made by the log-rank test.
Statistical significance was defined as a p value of less
than 0.05. All data analysis was performed by SAS Sys-
tem for Windows, version 8, and SAS Enterprise Guide
(SAS Institute, Cary, NC).
Between January 1999 and August 2003, a total of
519 patients underwent a kidney or combined
kidney-pancreas transplantation at our institution. Mean
follow-up was 884 Ϯ 23 days. Seventy-five patients had
at least one episode of ACR. On the basis of light
microscopic findings, 29 patients had AHR. The re-
cently developed consensus criteria for the diagnosis of
AHR were then used to confirm AHR . This en-
tailed C4d staining of frozen sections of transplant biopsy
samples and screening for donor-specific HLA antibod-
ies. C4d staining was performed on all 29 biopsy sam-
ples, and donor-specific HLA antibody screening was
performed on 23 (79%) of 29 patients. In 13 of 29
patients, donor-specific HLA antibody screening was
performed at the time of rejection. In 10 of 29 patients,
donor-specific HLA antibody screening was performed at
the time of transplantation. We defined AHR as pres-
ence of typical findings on light microscopy and presence
of either C4d staining or presence of donor-specific an-
tibodies. With this stringent approach, we were able to
confirm AHR in 23 patients and excluded the remaining
6 patients from the analysis (Figure 1).
The baseline characteristics of patients who developed
ACR, AHR, or No REJ are summarized in Table 1.
Although most demographic values did not differ be-
tween the groups, patients who experienced AHR were
significantly more likely to be black and female (AHR
vs. No REJ: p ϭ 0.0161 and p ϭ 0.0003, respectively).
Age and donor source were similar among groups.
Table 2 lists the clinical characteristics of our cohort.
Cold ischemia time was similar in all three groups.
Patients who developed AHR were significantly more
352 R.W. Lehrich et al.
likely to have had delayed graft function (AHR vs. No
REJ: p ϭ 0.0005). Use of induction therapy was similar
in all three groups. The time to rejection was defined as
the interval between renal transplantation and the diag-
nostic biopsy. Not surprisingly, AHR was diagnosed
earlier than ACR (median at day 6 vs. day 70, p ϭ
0.0071). However, two patients were found to have
AHR late in their transplant course, at days 147 and
843. Precipitating factors were unclear in the first pa-
tient but medication noncompliance was found to be the
cause in the second patient. All other patients in the
AHR group (21 of 23) experienced rejection between
days 3 and 14.
Patients in the AHR group had a significantly higher
mean T- and B-cell PRA compared with patients in the
ACR or No REJ group (mean B-cell PRA: AHR vs. No
REJ and ACR: p ϭ 0.0001 and p ϭ 0.0001, respectively;
mean T-cell PRA: AHR vs. No REJ and ACR: p ϭ
0.0001 and p ϭ 0.0002, respectively). When historic peak
PRAs were categorized as negative (Ͻ10%), moderate
(Ͼ10%–Ͻ50%), or high (Ͼ50%), we observed a bimodal
distribution of peak PRAs in the AHR group. Negative B-
and T-cell PRAs were found in 47.8% and 47.9% of
patients with AHR, respectively. High B- and T-cell
PRAs were identified in 43.5% and 47.9%, respectively.
The majority of patients in the ACR and No REJ group
were found to have a negative PRA (Figure 2).
The treatment of AHR consisted of PP and IVIG in
almost all patients (22 of 23). One patient received PP
alone. Eleven patients additionally received pulse meth-
ylprednisolone therapy, and seven patients received ei-
ther Thymoglobulin or OKT3 (Table 2). Most (20 of 23)
patients responded to therapy with improved renal func-
tion. Of the nonresponders, one patient required hemo-
dialysis on postoperative day 1 and underwent transplant
biopsy on day 6, the findings of which revealed AHR.
The patient was treated with methylprednisolone at that
point. PP and IVIG were initiated after a second biopsy
was performed on day 12, which revealed persistent
AHR. AHR could not be reversed, and the patient
continued to need renal replacement therapy. Transplant
nephrectomy was performed 7 months after the trans-
plant. The second patient was diagnosed on postopera-
tive day 2 with AHR and was treated with PP and IVIG
starting on day 3. The patient was discharged requiring
hemodialysis and died on postoperative day 30. The
TABLE 1 Demographic characteristics of patients who underwent renal
transplantation with and without acute rejection
(n ϭ 513)
(n ϭ 23)
(n ϭ 75)
(n ϭ 415)
mean Ϯ SD 46 Ϯ 0.6 45 Ϯ 2.6 42 Ϯ 1.5 47 Ϯ 0.6
Sex, n (%)
Male 302 (59%) 5 (22%) 45 (60%) 252 (61%)
Female 211 (41%) 18 (78%)a
30 (40%) 163 (39%)
Race, n (%)
White 292 (57%) 8 (35%) 31 (41%) 253 (61%)
Black 214 (42%) 15 (65%)b
44 (59%) 155 (37%)
Other 7 (1%) 0 0 7 (2%)
Type of transplant,
Living donor 197 (38%) 8 (35%) 24 (32%) 165 (40%)
transplant 316 (62%) 15 (65%) 51 (68%) 250 (60%)
p ϭ 0.0003 vs. No REJ.
p ϭ 0.0161 vs. No REJ.
FIGURE 1 Retrospective analysis of all consecutive kidney
and kidney-pancreas transplants performed at Duke University
Medical Center between January 1999 and August 2003.
Follow-up was extended until August 2004.
353IVIG and Plasmapheresis in Humoral Rejection
third patient was found to have AHR on postoperative
day 7; therapy with PP and IVIG was immediately
initiated. Subsequently, this patient developed systemic
inflammatory response with acute respiratory distress
syndrome, which was thought to be related to the epi-
sode of acute rejection. A transplant nephrectomy was
performed on postoperative day 13.
In the No REJ group, cumulative 2-year graft survival
was 94%, which was significantly higher than in both
rejection groups (ACR vs. No REJ: p Ͻ 0.0001; AHR
vs. No REJ: p ϭ 0.0002). Patients with ACR and AHR
had 2-year graft survival of 85% and 78%, respectively
(Figure 3). There was no significant difference in 2-year
graft survival between rejection groups (ACR vs. AHR:
p ϭ 0.50). Regarding patient survival, there was a sig-
nificant difference between patients in the ACR group
and No REJ (2-year patient survival: ACR 95% vs. No
REJ 98%, p ϭ 0.013). There were two deaths in the
AHR group (2-year patient survival: AHR 95%), but
mortality difference between the AHR group and No
REJ did not reach statistical significance (AHR vs. No
REJ: p ϭ 0.09) (Figure 4).. Last follow-up mean serum
creatinine of patients with functioning allografts for the
AHR, ACR, and No REJ groups were 1.8 mg/dl, 1.5
mg/dl, and 1.6 mg/dl, respectively (Table 3).
We report the results of a single-center retrospective
analysis of incidence and outcome of AHR in renal
transplantation. The central findings of this study are as
follows: (1) AHR occurs with an incidence of 4.4%,
affects predominantly highly sensitized patients, and is
observed early in the transplant course; (2) the combina-
tion of IVIG and PP is an effective strategy for the
treatment of AHR; and (3) 2-year graft survival of AHR
with this regimen is better than in historic controls and
comparable to graft survival in ACR.
When AHR was defined as allograft dysfunction with
typical light microscopic findings, as well as the presence
of positive C4d staining or of donor-specific antibodies,
the incidence of AHR in our study is comparable to
previous assessments of incidence and falls well into the
described range of 3%–10% [7, 24, 25]. The use of
evaluating allograft dysfunction for AHR with a com-
bined approach consisting of light microscopic evalua-
tion, immunofluorescence staining for C4d, and screen-
ing for donor-specific antibodies is now well established
and has been validated in several retrospective cohort
studies [7, 22, 24, 25]. We think that this approach
TABLE 2 Clinical characteristics of patients who underwent renal
Characteristic AHR (n ϭ 23) ACR (n ϭ 75) No REJ (n ϭ 415)
Cold ischemia time,
mean Ϯ SD 11.7 Ϯ 2.5 20.2 Ϯ 1.1 18.8 Ϯ 0.7
Delayed graft function,
n (%) 13 (56%)a
19 (25%) 90 (22%)
Induction therapy, n (%) 17 (74%) 43 (57%) 241 (58%)
Peak B-cell PRA,
mean Ϯ SD 39%bc
Ϯ 8.7% 7% Ϯ 2.5% 6% Ϯ 1.0%
Peak T-cell PRA 43%de
Ϯ 8.9% 9% Ϯ 2.9% 8% Ϯ 1.1%
Time to rejection,
median (IQR) 6f
(5–8) 70 (7–356) NA
Therapy for rejection, n (%)
PP ϩ IVIG 22 (96%) 0 NA
PP alone 1 (4%) 0 NA
IVIG alone 0 0 NA
Pulse methylprednisolone 13 (57%) 37 (49%) NA
Thymoglobulin or OKT3 7 (30%) 38 (51%)g
p ϭ 0.0005 AHR vs. No REJ.
p ϭ 0.0001 ACR vs. No REJ.
p ϭ 0.0001 AHR vs. ACR.
p ϭ 0.0001 AHR vs. No REJ.
p ϭ 0.0001 AHR vs. ACR.
p ϭ 0.0071 AHR vs. ACR.
In combination with pulse methylprednisolone in patients with ACR.
Cold ischemia time (hours); time to rejection (days).
354 R.W. Lehrich et al.
allowed us to reliably identify all patients with AHR to
retrospectively study the effectiveness of therapy with
IVIG and PP in AHR.
Patients in the AHR group had clinical features sim-
ilar to those previously described in patients with acute
alloantibody-mediated rejection. It is well established
that AHR occurs early in the transplant course, with a
median onset after transplantation measuring days rather
than weeks . The median onset of AHR in our study
was 6 days, with 50% of patients developing AHR
between days 5 and 8 after transplantation. However, we
were able to identify one patient who developed AHR
FIGURE 2 PRA frequencies according to PRA intensity.
Solid bars ϭ B-cell PRAs; open bars ϭ T-cell PRAs. PRAs
Ͻ10% were considered negative; PRAs 10%–50% were con-
sidered moderately elevated; and PRAs Ͼ50% were consid-
ered high. There were significantly more patients with high
PRAs in the AHR group compared with the ACR or the No
REJ group. *B-cell PRA: AHR vs. ACR and No REJ: p ϭ
0.0001 and p ϭ 0.0001, respectively. **T-cell PRA: AHR vs.
No REJ and ACR: p ϭ 0.0001 and p ϭ 0.0001, respectively.
FIGURE 3 Kaplan-Meier allograft survival curves with
groups as follows: No REJ group (solid line), ACR group
(dashed line), and AHR group (dotted line). Numbers above
x-axis at months 0, 6, 12, 18, 24, 30, and 36 represent number
of patients after censoring event. Cumulative graft survival was
significantly better in the No REJ group compared with ACR
and AHR (ACR vs. No REJ: pϽ 0.0001; AHR vs. No REJ: p
ϭ 0.0002). Graft survival between the AHR and ACR groups
was not significantly different (p ϭ 0.50).
FIGURE 4 Kaplan-Meier patient survival curves with
groups as follows: No REJ group (solid line), ACR group
(dashed line), and AHR group (dotted line). Numbers above
x-axis at months 0, 6, 12, 18, 24, 30, and 36 represent number
of patients after censoring event. Cumulative patient survival
was significantly better in the No REJ group compared with
ACR (ACR vs. No REJ: p ϭ 0.013). AHR patient survival was
not statistically different when compared with the ACR or the
No REJ group.
355IVIG and Plasmapheresis in Humoral Rejection
precipitated by medication noncompliance more than 2
years after transplantation. It is conceivable that sup-
pressed memory B cells were reactivated when immuno-
suppression was suboptimal, leading to late acute
alloantibody-mediated rejection. Highly sensitized pa-
tients are more likely to develop AHR [3, 7, 26].
Women in our cohort were significantly more likely to
develop AHR. This may relate to the higher rate of
sensitization observed in women as a result of previous
pregnancies. Last, elevated PRAs are markers of sensiti-
zation. It is therefore not surprising that patients in the
AHR group had significantly elevated historic peak
PRAs. However, PRA distribution was bimodal, and
roughly half of patients in the AHR group had a negative
PRA. Thus, a detectable PRA does not identify all
patients at risk for AHR. This emphasizes the notion
that donor-specific antibodies other than HLA antibod-
ies, and nonclassical HLA antibodies that are not de-
tected by the PRA method might play a role in AHR. In
cardiac transplantation, antiendothelial antibodies have
been demonstrated to be associated with acute humoral
but not ACR . In renal transplantation, antibodies to
MHC class I–related A antigen were found to be corre-
lated with rejection and early graft loss . The pres-
ence of activating antibodies to angiotensin II type 1
receptors was associated with steroid resistant rejec-
tion in a cohort of kidney transplant recipients who
also had malignant hypertension .
IVIG in combination with PP has been used by us and
others to treat AHR [10–13]. The commercial prepara-
tions of IVIG used in clinical practice contain intact IgG
molecules with a distribution of subclasses closely resem-
bling that in the human serum. IVIG represents pooled
plasma from approximately 3000–10,000 healthy do-
nors . A body of experimental and clinical evidence
suggests various potential actions of IVIG that might
explain its usefulness in treating AHR. In patients with
autoimmune hemophilia, IVIG has been demonstrated
to neutralize autoantibodies . This is likely because
of a high concentration of antiidiotypic antibodies in
IVIG directed against autoantibodies. In experimental
models of inflammatory activation of endothelial cells,
IVIG has been demonstrated to inhibit tumor necrosis
factor ␣– and interleukin 1␤–induced gene transcription
of adhesion molecules and cytokines [16, 29]. IVIG
likely behaves as normal IgG and IgM regarding the
control of autoreactivity of antibodies in human plasma
. Normal IgG and IgM function includes the sup-
pression of migration of B-cell populations from the bone
marrow to secondary lymphoid organs, as found in mice
. Furthermore, IVIG has been demonstrated to
downregulate specific autoreactive B-cell populations in
animal models of inherited immunodeficiency . It
can therefore be speculated that IVIG might have prop-
erties that are helpful in decreasing donor-specific anti-
body load and reducing harmful B-cell populations in
patients with AHR.
In our study, treatment of AHR with PP and IVIG is
associated with 2-year graft survival of 78%. Therapy
with PP alone is associated with inferior results [24, 33],
likely because of early rebound of alloantibodies. Historic
controls indicate that the graft loss without specific
therapy is 15%–50% [8, 9]. In our experience IVIG and
PP are helpful modalities to treat AHR, but our study
and similar studies by other groups must be interpreted
with caution. All conducted studies are retrospective.
Despite our encouraging results, graft loss in AHR re-
mains higher than in transplant recipients without re-
jection. New strategies involving alternative treatment
modalities are being investigated. Thymoglobulin in
combination with PP was recently used to treat renal
transplant patients with AHR. In a small, uncontrolled
study, no difference in graft survival between the AHR
group and the no rejection group was observed, making
this a promising treatment modality . Rituximab, a
genetically engineered chimeric human-murine anti–
CD-20 monoclonal antibody, has been used to treat
AHR. This approach appears reasonable because CD-20
is involved in the regulation of B-cell development and
differentiation. In two case reports (one heart transplant
recipient and one lung transplant recipient), rituximab
was a helpful adjunct in the treatment of AHR [35, 36].
TABLE 3 Outcome characteristics
Characteristic AHR (n ϭ 23) ACR (n ϭ 75) No REJ (n ϭ 415)
Follow-up (days) 764 Ϯ 109 944 Ϯ 58 880 Ϯ 26
2-year graft survival 78%a
2-year patient survival 95% 95%c
Last creatinine, median (IQR) 1.8 (1.4–2.6) 1.5 (1.2–1.9) 1.6 (1.3–1.8)
p ϭ 0.0002 AHR vs. No REJ.
p Ͻ 0.0001 ACR vs. No REJ.
p ϭ 0.013 ACR vs. No REJ.
2-year graft survival (%); 2-year patient survival (%); last creatinine, median (IQR) (mg/dl).
356 R.W. Lehrich et al.
Patients who are cross-match positive before transplan-
tation are at high risk of developing AHR. IVIG alone,
the combination of IVIG and PP, and the combination of
IVIG, PP, and rituximab have been used recently as part
of desensitization protocols in this patient population
[37–41]. These data support additional roles for IVIG
and PP, namely the prevention of AHR in high-risk
patients and overcoming contraindications for renal
In summary, we have demonstrated that the combi-
nation of IVIG and PP in addition to standard immu-
nosuppression containing prednisone, mycophenolate,
and calcineurin inhibition effectively salvages renal func-
tion in AHR. However, a higher rate of long-term graft
loss warrants more investigation into preventive and
R.W.L. is funded by a grant from the James R. Clapp Fellow-
ship in Nephrology.
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