AJCP / ORIGINAL ARTICLE

Correlation of Circulating Complement-Fixing Donor-Specific

Antibodies Identified by the C1q Assay and Presence of C4d
in Endomyocardial Biopsy Specimens
Renee Frank, MD,1 Priti Lal, MD,1 Jane Kearns, CHS,1 Maria R. Molina, MSN, CRNP,2
Joyce W. Wald, DO,2 Lee R. Goldberg, MD, MPH,2 and Malek Kamoun, MD, PhD1

From the 1Department of Pathology and Laboratory Medicine, and 2Heart Failure and Cardiac Transplant Program, Division of Cardiovascular Medicine,
University of Pennsylvania, Philadelphia, PA

Key Words: Endomyocardial biopsy; C4d; C1q assay; Donor-specific antibody

Am J Clin Pathol January 2016;145:62-68

DOI: 10.1093/AJCP/AQV016

ABSTRACT Donor-specific antibodies (DSAs) are associated with

Objectives: Donor-specific antibodies (DSAs) are associated
with increased cardiac graft loss. We applied a C1q solid-
phase assay in parallel with the standard immunoglobulin G
(IgG) single antigen bead (SAB) assay to examine the correl-
ation of circulating complement-fixing donor-specific antibod-
ies and the presence of C4d in endomyocardial biopsy (EMB)
specimens.
Methods: We retrospectively studied the relationship of C1qþ
DSAs and C4d immunofluorescence (IF) in 49 EMB specimens
from 44 heart transplant recipients who had concurrent EMB,
C4d IF, and DSA measurements. We applied a C1q SAB in
parallel with the standard IgG SAB assay to examine the DSA
profiles in heart transplant patients posttransplant.
Results: A better concordance is observed between C1qþ
DSAs with C4d IFþ compared with IgG DSAs with C4d
IF þ (40% vs 24%, P ¼ .02). However, the correlation of C1q
DSAs with C4d IF is not statistically significant (P ¼ .24).
Importantly, C1qþ DSAs were observed in 16 of 17 cases
with C4d IFþ; 24 cases had circulating C1qþ DSAs without
detectable C4d staining, suggesting that that the presence of
C1qþ DSAs may precede the detection of C4d deposition in
EMB specimens and/or the development of antibody-medi-
ated rejection.
Conclusions: In this cohort of 44 patients, no significant
correlation was observed between circulating C1q DSAs
and C4d IF in EMB specimens. Additional studies are
needed to further evaluate the association of C1q DSAs with
EMB specimens and C4d staining.
increased cardiac allograft injury and loss, including vascular
injury and graft dysfunction. Detection of antibody-mediated
rejection (AMR) relies in part on graft dysfunction, endomyo-
cardial biopsy (EMB) histopathologic criteria, and circulating
DSAs.1,2 DSAs can be against anti–class I human leukocyte
antigen (HLA), anti–class II HLA, or non–anti-HLA antibod-
ies.3 Since Patel and Terasaki4 demonstrated a significant cor-
relation between a positive crossmatch and hyperacute and
accelerated acute grafts dysfunction in kidney transplants using
a complement-dependent cytotoxicity (CDC) assay in 1969,
there has been no highly sensitive and specific assay predictive
of clinical outcomes in transplantation patients. The currently
available assays for detection of DSAs uses either CDC, or
flow cytometry or Luminex (Luminex Corporation, Austin TX)
technology using beads coated with single HLA antigens.
While these assays are highly sensitive, they fail to differentiate
antibodies that fix complement and cause graft injury or dys-
function from antibodies that do not cause graft injury or dys-
function. In the end, it is the pathogenicity, or ability of the
antibody to fix complement, that is most pertinent to the acute
and chronic effects of DSAs and ultimately clinical outcome.5-8
Detection of antibodies capable of binding complement,
specifically antibodies binding the first component of the
classic pathway, C1q, gives the earliest indication of the po-
tential for complement-mediated injury.9,10 A C1q single
antigen bead (SAB) assay to detect complement-fixing HLA
antibodies has recently been developed.9,11 This assay is
highly sensitive and specific for distinguishing subsets of im-
munoglobulin G (IgG) antibodies that can fix complement
and assess risk of early clinical AMR in cardiac

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allografts.12,13 A strong correlation between preformed com-
plement-fixing DSAs detected by a C1q SAB, positive CDC
crossmatch, and early clinical AMR has also been shown.12,13
Interestingly, in the kidney transplantation literature, comple-
ment-fixing DSAs detected by a C1q SAB fixation and C4d
deposition in kidney transplant biopsy specimens do not ap-
pear to be directly correlated.14
In the cardiac transplant literature, studies have eval-
uated early clinical AMR with C4d staining of EMB speci-
mens and complement-fixing DSAs detected by a C1q SAB
assay; however, to our knowledge, there are no systematic
studies evaluating DSAs detected by a C1q SAB with C4d
staining and other parameters, including demographics and
comorbidity factors.15,16 We applied a C1q SAB in parallel
with the standard IgG SAB to examine the correlation of
circulating complement-fixing donor-specific antibodies
and presence of C4d in EMB specimens.
Materials and Methods
Patient Population
In this retrospective analysis, we studied the relationship
of complement-fixing DSAs and C4d immunofluorescence
(IF) in 40 EMB specimens from 38 cases of previously
selected heart transplant recipients.17 Heart transplant patients
(2005-2011) with concurrent endomyocardial biopsy, im-
munofluorescence for C4d, and DSA measurements were
initially selected. Thirty-eight patients had stored serum sam-
ples available for this analysis. Clinical data gathered included
patient demographics, presence and HLA class specificity of
DSAs, ejection fraction (EF), nonimmunologic comorbidities,
and cause of death. Nonimmunologic comorbidities gathered
from the electronic medical record included presence or
absence of hypertension, diabetes mellitus, cytomegalovirus
(CMV) viremia, obesity, and hyperlipidemia.18-22 CMV vir-
emia was defined as symptomatic patients with a quantitative
CMV viral load of more than 5,000 copies/mL who were
treated with intravenous (IV) ganciclovir. Allograft dysfunc-
tion was defined as a precipitous decline in EF by at least 10%
or an EF 35% or less, detected by transthoracic echocardio-
gram or transesophageal echocardiogram. To be included in
the cohort, the measurement of EF was done within 61 month
of the EMB. Cardiovascular mortality was defined by death
due to acute AMR, acute myocardial infarction, and progres-
sive allograft dysfunction and heart failure. For the purposes of
this study, patients who died of noncardiac causes were cen-
sored from the graft failure analysis. Official cause of death
was obtained from our electronic medical record systems,
which were recorded directly from the patient death certificate
and autopsy report when applicable. The maintenance im-
munosuppression protocols, including a calcineurin inhibitor,
© American Society for Clinical Pathology
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AJCP / ORIGINAL ARTICLE
antimetabolite, or a cell cycle inhibitor, have been described in
our previous study.17 At our institution, all patients are on
maintenance immunosuppression protocols, including a calci-
neurin inhibitor, antimetabolite, or a cell cycle inhibitor. The
target tacrolimus level is 10 to 15 lg/L at 0 to 6 months, 10 to
12 lg/L at 6 to 12 months, 8 to 10 lg/L at 12 to 24 months,
and 5 to 8 lg/L at more than 24 months. The target cyclospor-
ine level is 200 to 300 ng/mL at 1 to 6 months, 150 to 200 ng/
mL at 6 to 12 months, 120 to 175 ng/mL at 12 to 24 months,
and 80 to 100 ng/mL at more than 24 months. All patients who
exhibit acute cellular rejection (ACR) grade 2R are treated
with IV solumedrol, 1 g, for 3 days if the patient is status
posttransplant less than 6 months, or prednisone, 100 mg, for 5
days if the patient is status posttransplant more than 6 months.
If the patient is exhibiting ACR grade 2R or 3R with hemo-
dynamic compromise and a decrease in EF of more than 10%,
then the patient will also be treated with received rabbit anti–
thymocyte globulin (thymoglobulin). There was no difference
in treatment since all treatments were similar for each patient,
with the exception of plasmapheresis and photopheresis.
Access to patient medical record information and review of
archived myocardial biopsy tissue was granted approval by the
Hospital of the University of Pennsylvania Institutional
Review Board.
Endomyocardial IF
IF for C4d is performed on one to three pieces of fresh tis-
sue submitted by the clinical team in addition to tissue for rou-
tine H&E evaluation. C4d IF staining is not performed in our
institution within the first 2 weeks of transplantation. In gen-
eral, fresh-frozen tissue was analyzed by indirect IF micros-
copy technique by using antibodies directed against C4d
primary affinity-purified monoclonal antibody (dilution, 1:50;
30-minute incubation at room temperature; Sigma Aldrich, St
Louis, MO). The fresh-frozen tissues were cut at 3 to 4 lm
thickness and mounted on single-ring positively charged slides
(Fisher, Pittsburgh, PA). Staining was performed according to
standard procedures. Indirect IF microscopy was performed on
a Nikon OPTISHOT with a Nikon Coolscan V ED camera
(Nikon, Melville, NY) using Image Pro Plus (Media
Cybernetics, Rockville, MD). Interstitial capillary C4d staining
was semiquantitatively graded on a scale of 0 to 3þ for inten-
sity, and a percentage of interstitial capillary staining over the
entire parenchyma was assigned. Any EMB specimen exhibit-
ing at least 2þ C4d staining over 50% of the interstitial capilla-
ries was considered significant or positive for C4d deposition,
an example of which is shown as Image 1 . In this study,
AMR was defined as clinical evidence of graft dysfunction,
along with positive DSA and IF C4d (as defined above). C4d
evaluation is performed at the request of the clinical team in
(1) patients with clinical evidence of graft dysfunction (new
Am J Clin Pathol 2016;145:62-68 63
DOI: 10.1093/ajcp/aqv016
 Frank et al / DSAS BY C1Q ASSAY WITH C4D IN EMB SPECIMENS

heart failure symptoms, new S3 murmur, increased jugular
venous distention, and a decrease in EF of >10%), (2) patients
who exhibit high percent reactive antibody, and (3) all patients
with ACR of International Society for Heart and Lung
Transplantation grade 3R. Most currently, AMR has been pro-
posed to be a diagnosis rendered by pathologists, without the
requirements of clinical dysfunction or positive DSAs, which
was required in the past.3,23,24 It must be emphasized that the
pathologic AMR (pAMR) grading system represents an initial
guideline with the intention of accumulating data, validation,
and patient treatment regimens. Furthermore, the recognition
and diagnosis of AMR by histopathology, even among experi-
enced transplant pathologists, is challenging. A number of
studies have highlighted the problems of key morphologic
criteria for the purposes of AMR screening.25,26 The threshold
for therapeutic intervention in patients with AMR remains un-
known, particularly in the setting of “asymptomatic AMR.”
For these reasons, we have not used the pAMR grade to evalu-
ate the utility of the C1q assay.
HLA Typing and Evaluation of Anti-HLA Antibodies
HLA typing was performed on all donors and recipients
using DNA-based methods. Initial HLA typing included
HLA-A, B, C, DRB1, DRB3/4/5, and DQB1. For recipients
with antibodies against HLA-DQA, DP, or allele specific
antibodies, additional typing of these loci for donors and re-
cipients was also performed on a subset of these patients.
Identification of anti-HLA antibodies was performed using

Image 1 Endomyocardial biopsy specimen exhibiting at least 2þ C4d staining over 50% of the interstitial capillaries, con-
sidered positive for C4d deposition. A, Unremarkable myocardium (H&E, 5). B, Myocardium with diffuse endothelial cell pro-
liferation (arrows) and interstitial myxoid change (arrowheads) (H&E, 5). C, C4d immunofluorescence in interstitial capillaries.

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solid-phase class I and class II assays, which included flow Table 1

cytometry bead and/or Luminex bead assays. At least two Clinical Information for Patients With C1q Donor-Specific
Antibodies and Those Without C1q Donor-Specific Antibodies
antibody solid-phase testing methods were used, one of which
With Nonimmunologic Comorbidities of Patients With and
was a mixed-bead class I and class II assay or a class I and Without DSAsa
class II phenotype assay. HLA specificities were confirmed
C1q DSAs
by single-antigen bead assays (One Lambda, Canoga Park,
CA). Evaluation of complement-binding anti-HLA antibodies Characteristic Positive (n ¼ 40) Negative (n ¼ 9)
was detected by using C1q single antigen bead assays (One Sex
Lambda). For sensitized patients, antibody testing included Male 31 6
HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, and DPB1 Female 9 3
Age at time of biopsy, mean 48 (21-78) 43 (22-63)
specificities. A retrospective or prospective flow cytometric (range), y
crossmatch was performed on all patients. The strength of Pretransplant DSAs 5 3
antibody determined using solid-phase assays was correlated Concurrent episode of ACR 36 8
Etiology of heart failure
with the crossmatch test results. Donor-specific anti-HLA
Ischemic CM 10 3
antibodies were considered positive if the median fluores- Dilated CM 12 1
cence intensity assessed by Luminex single antigen bead Hypertrophic CM 2 0
assays was greater than 1,000 for IgG and higher than 400 for Congenital CM 1 0
Restrictive CM 1 0
the C1q assays. Nonischemic CM NOS 14 5
Nonimmunologic comorbidities
Statistical Analysis CMV viremia 7 1
Hyperlipidemia 40 8
Statistical analysis was performed using Fisher’s exact t Obesity (BMI >30 kg/m2) 6 1
test and a paired samples t test. Statistical significance was Diabetes mellitus 24 6
defined as P < .05 (MedCalc, version 9.3.0.0 (MedCalc Hypertension 37 9

Software, Ostend, Belgium)). ACR, acute cellular rejection; BMI, body mass index; CM, cardiomyopathy; CMV,
cytomegalovirus; DSAs, donor-specific antibodies; NOS, not otherwise specified.
a
Values are presented as numbers unless otherwise indicated.

Results
Patient Characteristics
Upon review of our pathology archives between 2005
and 2011, a total of 109 patients had concurrent EMB, DSA
evaluation, and C4d IF.17 Of these 109 patients, 44 had
stored serum samples for C1q SAB analysis. The current co-
hort comprises 49 allografts from 44 patients, including 11
women and 33 men. Patient demographics and characteris-
tics are shown in Table 1 . Four patients received a second
cardiac transplant that was included in the study group. Of
our patient cohort, four were treated with photopheresis and
five were treated with a combination of plasmapheresis
(courses of four to six exchanges), IV immunoglobulin, rit-
uximab, and cytoxan.
Histopathology
We analyzed a total of 49 EMB and concurrent IF stud-
ies from 49 cardiac grafts in 44 patients who had pre- and
posttransplant DSA measurements. Mild and moderate acute
cellular rejection was seen in 32 and 12 grafts, respectively.
There were no EMB specimens with severe acute cellular re-
jection in this cohort. As shown in Table 1, nonimmunologic
comorbidities of the C1q DSA and non-C1q DSA patient
groups are similar. The distribution of total IgG DSAs and
C1qþ DSAs is shown in Figure 1 ; 84% (36/43) of patients
who had IgG DSAs had C1qþ complement-fixing DSAs in
this selected cohort of patients. Importantly, C1qþ DSAs
were observed in 16 of 17 cases with C4d IFþ; 24 cases had
circulating C1qþ DSAs without detectable C4d staining
Table 2 . However, the correlation of C1q DSAs with C4d
IF is not statistically significant (P ¼ .24).
EMB Characteristics and Graft Dysfunction
EMB IF results were compared with corresponding
graft EFs. Of the 49 biopsies, 28 (70%) EMBs were per-
formed during a clinically significant decline in EF as
defined above. Twenty-three (82%) of the 28 EMB speci-
mens evaluated during an episode of graft dysfunction cor-
related with positive C1q DSAs. As shown in Table 3 , in
patients with positive C1q DSAs (n ¼ 23), positive C4d
staining on the EMB specimen was observed in 13 (56%).
In addition, in patients who were tested negative for C1q
DSAs, positive C4d staining on the EMB specimen was
observed in only one (20%).
Graft Failure
In this study group of a total of 44 patients with 49 grafts
evaluated, 18 patients died or underwent a heart retransplant.
Two patients were not included in the graft failure analysis

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since one patient was lost to follow-up and one did not meet
cardiac mortality criteria (died secondary to acute myeloid leu-
kemia). After these two exclusions, 47 allografts were eval-
uated for graft failure. As shown in Table 4 , overall, 38%
(18/47) cardiac allografts failed. Graft failure occurred in 14
(37%) of 38 patients who tested positive for C1q DSAs and in
four (44%) of nine patients who tested negative for C1q DSAs;
three of these four patients developed IgG DSAs. The mean
time to graft failure was 56 months and 59 months for grafts
with and without C1qþ DSAs, respectively. Of the 28 allo-
grafts that survived, 24 (86%) tested positive for C1q DSAs.
Similarly, 83% (15/18) of allografts that failed tested positive
for C1q DSAs.

20 Discussion
No. of Cardiac Allografts

18 C1q+
16 C1q– In this report, we present a single-institution study of a
14
subset of 44 patients who underwent cardiac transplants and
12
10 had comprehensive clinical, pathologic, and immunologic
8 data to study the relationship of these parameters with cardiac
6
4 allograft transplantation outcomes. Our data suggest no sig-
2 nificant correlation between circulating complement-fixing
0
No DSA DSA to HLA DSA to HLA DSA to Both
DSAs detected by C1q SAB and C4d IF in EMB specimens.
(n = 6) Class I Class II HLA Class A better concordance is observed between C1qþ DSAs with
(n = 5) (n = 19) I and II
(n = 19) C4d IF þ compared with total IgG DSAs with C4d
IF þ (40% vs 24%, P ¼ .02).
Figure 1 Donor-specific antibodies by human leukocyte
Patients with concurrent data for graft dysfunction, EMB,
antigen (HLA) class as related to C1q positivity.
IF for C4d, and C1q DSA were analyzed. Nonimmunologic
comorbidities were also evaluated to overcome possible con-
Table 2
C4d Immunofluorescence With and Without C1q Donor- founding factors, and were similar in patients who had circulat-
Specific Antibodiesa ing C1qþ DSAs and those who remained negative for C1q
DSAs (Table 1). However, the correlation of C1q DSAs with
C1q DSAs, No. (%)
C4d IF was not statistically significant (P ¼ .24). Importantly,
Characteristic Positive (n ¼ 40) Negative (n ¼ 9) Total No. C1qþ DSAs were observed in 16 of 17 cases with C4d IFþ;
C4d positive 16 (40) 1 (11) 16 24 cases had circulating C1qþ DSAs without detectable C4d
C4d negative 24 (60) 8 (89) 32 staining (Table 2), suggesting that the presence of C1qþ
Total 40 (100) 9 (100) 49
DSAs may precede the detection of C4d deposition in EMB
DSAs, donor-specific antibodies. specimens and/or the development of AMR. In this cohort of
a
The correlation of C1q DSAs with C4d immunofluorescence is not statistically sig-
nificant (P ¼ .24).
38 patients, no significant correlation was observed between
circulating C1q DSAs and C4d IF in EMB specimens. In pa-
tients with graft dysfunction, 82% had circulating C1q DSAs.
Table 3 There was no significant correlation observed between circu-
Endomyocardial Biopsy During an Episode of Allograft lating C1q DSAs and graft failure; this lack of correlation may
Dysfunction With Corresponding C4d Immunofluorescence in part be due to a patient selection bias since our cohort was
Staining (P ¼ .619) focused on patients who had a graft dysfunction, EMB, and IF
C1q DSAs No C4d, No. (%) C4d >50%, No. (%) C4d staining.
The clinical utility of novel assays detecting complement
Positive (n ¼ 23) 13 (56) 10 (44)
Negative (n ¼ 5) 4 (80) 1 (20) binding anti-HLA antibodies was recently reported in heart
transplantation.12,15 However, the role of complement-fixing

Table 4
Cardiac Allograft Failure With Corresponding C1q DSA Status and C4d Immunofluorescence Staining (P ¼ .716)
Graft Failure, Time to Graft Failure, Negative C4d, No./ Positive C4d, No./ IgG DSAs, No./
C1q DSAs No. (%) Mean (Range), mo Total No. (%) Total No. (%) Total No. (%)

Positive (n ¼ 38)a 14 (37) 56 (9-162) 9/14 (64) 5/14 (36) 14/14 (100)
Negative (n ¼ 9) 4 (44) 59 (17-139) 4/4 (100) 0 3/4 (75)

DSAs, donor-specific antibodies; IgG, immunoglobulin G.

a
Two patients censored from graft failure analysis: one patient died of acute myeloid leukemia, and the second patient was lost to follow-up.

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vs non–complement-fixing DSAs and its correlation with C4d
IF staining had not yet been elucidated. All DSAs are not equal
in terms of detriment to allograft function.27 The ability to acti-
vate the complement cascade appears to be a critical factor in
differentiating clinically relevant and nonrelevant DSAs.12 A
positive C1q DSA with a negative C4d staining in EMB speci-
mens could be due to a number of factors, including low sensi-
tivity of IF staining, low sensitivity of the threshold used for a
positive read on IF, or lack of C3d testing in our cohort. It
should also be noted that a patient could have positive C4d
staining without circulating DSAs secondary to non-HLA
antiallograft antibodies or due to complement fixation second-
ary to infections or other types of injuries.28
In this cohort of 44 patients, no significant correlation
was observed between circulating C1q DSAs and C4d IF in
EMB specimens. Prospective studies are needed to further
evaluate the association of C1q DSAs with EMB and IF
C4d staining, as well as with long-term outcomes in heart
transplant recipients. Because our study is limited to a
small cohort, additional studies of the impact of treatment
strategies of AMR with circulating C1qþ DSAs are neces-
sary to transform the diagnostic data into an actionable
treatment plan that improves long-term heart transplant
outcomes.
Corresponding author: Renee Frank, MD, University of
Pennsylvania, 3400 Spruce St, 6th Floor, Founders Building,
Philadelphia, PA 19104; renee.frank@uphs.upenn.edu.
We thank Insuk Choe, JD, for help in compiling and in the crit-
ical evaluation of HLA data. We appreciate the assistance of
Lynita Thomas and Christopher Mignogna with the IF processing
protocols.
These data were presented in part at the American Society for
Histocompatibility and Immunogenetics, 39th Annual Meeting,
Chicago, IL, November 17-21, 2013.
References
1. Kobashigawa J, Crespo-Leiro MG, Ensminger SM, et al.
Report from a consensus conference on antibody-mediated re-
jection in heart transplantation. J Heart Lung Transplant.
2011;30:252-269.
2. Stewart S, Winters GL, Fishbein MC, et al. Revision of the
1990 working formulation for the standardization of nomen-
clature in the diagnosis of heart rejection. J Heart Lung
Transplant. 2005;24:1710-1720.
3. Berry GJ, Angelini A, Burke MM, et al. The ISHLT work-
ing formulation for pathologic diagnosis of antibody-medi-
ated rejection in heart transplantation: evolution and
current status (2005-2011). J Heart Lung Transplant.
2011;30:601-611.
4. Kfoury AG, Stehlik J, Renlund DG, et al. Impact of repetitive
episodes of antibody-mediated or cellular rejection on
cardiovascular mortality in cardiac transplant recipients:
defining rejection patterns. J Heart Lung Transplant.
2006;25:1277-1282.
© American Society for Clinical Pathology
Downloaded 67
from https://academic.oup.com/ajcp/article-abstract/145/1/62/1766923
by guest
on 17 February 2018
AJCP / ORIGINAL ARTICLE
5. Bohmig GA, Exner M, Watschinger B, et al. C4d deposits in
renal allografts are associated with inferior graft outcome.
Transplant Proc. 2001;33:1151-1152.
6. Shahzad K, Aziz QA, Leva JP, et al. New-onset graft dysfunc-
tion after heart transplantation-incidence and mechanism-
related outcomes. J Heart Lung Transplant. 2011;30:194-203.
7. Colvin RB, Smith RN. Antibody-mediated organ-allograft re-
jection. Nat Rev Immunol. 2005;5:807-817.
8. Tan CD, Sokos GG, Pidwell DJ, et al. Correlation of donor-
specific antibodies, complement and its regulators with graft
dysfunction in cardiac antibody-mediated rejection. Am J
Transplant. 2009;9:2075-2084.
9. Herskowitz A, Soule LM, Ueda K, et al. Arteriolar vasculitis
on endomyocardial biopsy: a histologic predictor of poor out-
come in cyclosporine-treated heart transplant recipients. J
Heart Transplant. 1987;6:127-136.
10. Kfoury AG, Hammond ME, Snow GL, et al. Cardiovascular
mortality among heart transplant recipients with asymptom-
atic antibody-mediated or stable mixed cellular and antibody-
mediated rejection. J Heart Lung Transplant. 2009;28:781-784.
11. Takemoto SK, Zeevi A, Feng S, et al. National conference to
assess antibody-mediated rejection in solid organ transplant-
ation. Am J Transplant. 2004;4:1033-1041.
12. Baldwin WM, Kasper EK III, et al. Beyond C4d: other comple-
ment-related diagnostic approaches to antibody-mediated re-
jection. Am J Transplant. 2004;4:311-318.
13. Mehra MR, Crespo-Leiro MG, Dipchand A, et al.
International Society for Heart and Lung Transplantation
working formulation of a standardized nomenclature for car-
diac allograft vasculopathy—2010. J Heart Lung Transplant.
2010;29:717-727.
14. Yabu JM, Higgins JP, Chen G, et al. C1q-fixing human leuko-
cyte antigen antibodies are specific for predicting transplant
glomerulopathy and late graft failure after kidney transplant-
ation. Transplantation. 2011;91:342-347.
15. Chin C, Chen G, Sequeria F, et al. Clinical usefulness of a
novel C1q assay to detect immunoglobulin G antibodies
capable of fixing complement in sensitized pediatric heart
transplant patients. J Heart Lung Transplant. 2011;30:158-
163.
16. Zeevi A, Lunz J, Feingold B, et al. Persistent strong anti-
HLA antibody at high titer is complement binding and asso-
ciated with increased risk of antibody-mediated rejection in
heart transplant recipients. J Heart Lung Transplant.
2013;32:98-105.
17. Frank R, Molina MR, Wald JW, et al. Correlation of circulat-
ing donor-specific anti-HLA antibodies and presence of C4d
in endomyocardial biopsy with heart allograft outcomes: a sin-
gle-center, retrospective study. J Heart Lung Transplant.
2013;32:410-417.
18. Braga JR, Santos IS, McDonald M, et al. Factors associated
with the development of cardiac allograft vasculopathy—a sys-
tematic review of observational studies. Clin Transplant.
2012;26:E111-E124.
19. Chen W, Thoburn CJ, Miura Y, et al. Autoimmune-mediated
vasculopathy. Clin Immunol. 2001;100:57-70.
20. de Denus S, Al-Jazairi A, Loh E, et al. Dyslipidemias
and HMG-CoA reductase inhibitor prescription in heart
transplant recipients. Ann Pharmacother. 2004;38:1136-1141.
21. Kahn J, Rehak P, Schweiger M, et al. The impact of over-
weight on the development of diabetes after heart transplant-
ation. Clin Transplant. 2006;20:62-66.
Am J Clin Pathol 2016;145:62-68 67
DOI: 10.1093/ajcp/aqv016
 Frank et al / DSAS BY C1Q ASSAY WITH C4D IN EMB SPECIMENS

22. McDonald K, Rector TS, Braulin EA, et al. Association of cor-
onary artery disease in cardiac transplant recipients with cyto-
megalovirus infection. Am J Cardiol. 1989;64:359-362.
23. Berry GJ, Burke MM, Andersen C, et al. The 2013 International
Society for Heart and Lung Transplantation Working Formulation
for the standardization of nomenclature in the pathologic diagnosis
of antibody-mediated rejection in heart transplantation. J Heart
Lung Transplant. 2013;32:1147-1162.
24. Reed EF, Demetris AJ, Hammond E, et al. Acute antibody-
mediated rejection of cardiac transplants. J Heart Lung
Transplant. 2006;25:153-159.
25. Fedrigo M, Gambino A, Benazzi E, et al. Role of morphologic
parameters on endomyocardial biopsy to detect sub-clinical
antibody-mediated rejection in heart transplantation. J Heart
Lung Transplant. 2011;30:1381-1388.
26. Hammond ME, Stehlik J, Snow G, et al. Utility of histologic
parameters in screening for antibody-mediated rejection of the
cardiac allograft: a study of 3,170 biopsies. J Heart Lung
Transplant. 2005;24:2015-2021.
27. Stoica SC, Cafferty F, Pauriah M, et al. The cumulative effect
of acute rejection on development of cardiac allograft vascul-
opathy. J Heart Lung Transplant. 2006;25:420-425.
28. Amico P, Honger G, Bielmann D, et al. Incidence and
prediction of early antibody-mediated rejection due to non-
human leukocyte antigen-antibodies. Transplantation.
2008;85:1557-1563.

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