Clinical Relevance of Complement Fixing Antibodies in Cardiac
Marlene L. Rose, John D. Smith
Reference: HIM 8233
To appear in: Human Immunology
Received date: 25 February 2009
Accepted date: 9 April 2009
Please cite this article as: Rose, M.L., Smith, J.D., Clinical Relevance of Complement
Fixing Antibodies in Cardiac Transplantation, Human Immunology (2009), doi:
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Clinical Relevance of Complement Fixing Antibodies in Cardiac Transplantation
Marlene L.Rose1 and John D. Smith2
National Heart and Lung Institute, Imperial College1, Transplant Immunology1,2
Harefield Hospital, Harefield, Middlesex, UK
Corresponding author: Marlene Rose, Imperial College, Heart Science Centre,
Harefield Hospital, Harefield, Middlesex, UB9 6JH UK.
Key words: Cardiac Transplantation, Alloantibodies, non-HLA antibodies,
Complement, HLA antibodies
Abbreviated title: Antibodies in cardiac transplantation
In this review, the relative importance of pre-transplant complement fixing and non-
complementing fixing HLA and non-HLA antibodies is reviewed. Sera from 565
adult cardiac transplant recipients was retrospectively analysed for the presence of
HLA antibodies using CDC, HLA coated Luminex beads and C4d deposition on
Luminex beads and results correlated with graft survival. Whereas 14/565 patients
had CDC positive donor specific antibodies (DSA) before their transplant, this
number was increased by 53 using Luminex beads, of these 19 had DSA by Luminex.
Patients negative for CDC cross-match, but Luminex +ve with DSA had poor one
year survival (42%) compared to 77% one–year survival of patients with CDC-ve,
Luminex +ve non-donor specific antibodies. The effect of donor specific antibodies
on allograft survival was further analysed according to titre, immunoglobulin sub –
class and ability to fix C4d onto Luminex beads. Ability to fix C4d, but not antibody
titre or Ig subclass was strongly associated with poor allograft survival (P=0.0002). A
retrospective of sera form 616 cardiac transplant patients revealed presence of IgM
complement fixing non-HLA antibodies was associated with early graft failure and a
diagnosis of Primary Graft Failure. In conclusion, complement fixing antibodies to
relevant antigens are associated with poor allograft survival after heart transplantation.
It is well established that transplantation of hearts into patients with pre-formed donor
specific complement fixing HLA antibodies results in hyperacute or accelerated acute
rejection , in much the same was as would happen if kidneys were transplanted into
sensitised recipients . Until about 1995, the cytotoxic cross match, which measures
complement fixing antibodies was used to determine whether patients had antibodies
to donor leukocytes. Since that time there have been major advances in the technology
used to detect antibodies, and a greater understanding of importance of non-HLA
antibodies in causing early damage to the graft. The use of solid phase assays has
increased sensitivity such that approximately 5 times as many patients make HLA
antibodies compared to the cytotoxic method . The increased sensitivity raises the
question about the clinical relevance of these antibodies, since many of the antibodies
detected will not be complement fixing; further stratification of antibodies may be
necessary to avoid depriving patients of transplants due to antibodies which may not
be important. The purpose of this review is to summarise about what is known about
the clinical relevance of pre-transplant antibodies detected by solid phase assays. In
addition the role of complement fixing non-HLA antibodies will be reviewed.
a) HLA antibodies.
Studies from renal centres have demonstrated that presence of pre-transplant donor
specific HLA antibodies detected by solid phase assays [4,5], in the absence of a
positive cross-match with donor cells, are associated with higher rates of graft failure.
In order to investigate whether this was the case for heart transplant recipients, we
undertook a large retrospective study of pre-transplant serum from 565 adult cardiac
transplant recipients transplanted between 1991-1995. All patients had been
transplanted with a negative cross-match to donor T cells, except 4 who were
transplanted prior to 1992 and a recent patient who become HLA antibody positive
due to a unreported pregnancy. All sera were tested for HLA antibody reactivity using
standard complement dependent cytoxicity (CDC) assays and solid phase Luminex
based assays. Patients were initially screened using Luminex multiple antigen beads;
positive sera was further analysed for HLA specificity using single antigen beads, and
donor specificity was assigned. Prior to transplantation 14 patients were known to
have produced HLA antibodies, detectable by CDC assays. Retrospective analysis
using the Luminex assays revealed a further 53 patient samples contained HLA
antibodies. CDC assays detected HLA class I antibodies in 12 serum compared with
54 using Luminex and class II antibodies in 4 compared with 25 by Luminex (Table
1). All sera that were positive by CDC were also positive by the Luminex assay
(Table 1). Actuarial graft survival was analysed according to pre-transplant HLA
antibody status (Fig 1); it can be seen that the 5 patients transplanted with CDC+ve
cross-match and Luminex +ve donor specific antibodies (DSA) showed poor one year
survival (41.7%). Similarly, patients negative for CDC cross-match, but Luminex +ve
with DSA also had poor one year survival (42%) compared to 77% one–year survival
of patients with CDC-ve, Luminex +ve non-donor specific antibodies. The survival of
patients with non-donor reactive HLA antibodies, detected by Luminex, is equivalent
to patients negative for antibodies (Fig 1). These results attest to the importance of
donor specific HLA antibodies as determinant of allograft survival. The majority of
graft failure within the donor specific groups occurred within the first 3 months after
Properties of HLA Abs that influence pathogenecity
Little is known about the properties of antibodies that contribute to their
pathogenecity in a transplant setting. Factors likely to be important include, ability to
fix complement, titre, immunoglobulin subclass and antigen specificity.
C4d fixing antibodies: It is known that antibody mediated activation of complement
and deposition of various split components (C1q, C4b, C4d, C3a, C3d) contribute to
graft damage in the absence of assembly of the terminal lytic complex (C5-C9) and
cell lysis[6,7]. Thus deposition of C4d along peritubular capillaries of renal biopsies
 and to a lesser extent in cardiac biopsies  has become a useful adjunct to
diagnosis of acute humoral rejection. We therefore devised an assay to determine
ability of Luminex binding antibodies to fix C4d on to Luminex beads ]. Pre-
transplant serum from sixty-seven patients (from the 565 patients in the 1991-2005
study described above) were found to contain HLA abs detected by Luminex, of these
22 cause C4d deposition on Luminex beads. Of the 22 Luminex positive C4d fixing
sera, 11 contained DSA and 11 had no detectable DSA. The one year graft survival
for C4d+ DSA (n=11) was 20% compared to 91% for C4d+ve non-donor specific abs
(n=11), 54% for C4d-ve DSA (n=13) and 75% for antibody negative recipients
(p=0.0002). Fig 2. These results demonstrate that pre-transplant DSA that fix C4d in
this test, are as poor a prognostic indicator as an IgG cytotoxic cross-match. It is
interesting that graft survival of patients with DSA that do not fix C4d is in between
those who are antibody negative and those with C4d fixing DSA. It may be that pre-
transplant antibodies which do not fix C4d become complement fixing after
transplantation. On the other hand, antibodies that do not fix C’ may be damaging due
to their ability to activate endothelial cells  and cause exocytosis of weibel palade
bodies and hence producing a pro-coagulation phenotype .
Antibody Titre; An analysis of 145 serum samples containing HLA antibodies as
defined by Luminex assays was performed comparing Mean Fluorescent Intensity
(MFI) values to C4d binding results. The mean MFI of the highest bead value in the
51 serum which demonstrated C4d fixation in the Luminex assay was 13069 +/- 5200,
compared to 4982 +/- 3335 for the C4d negative group p<0.0001. However, 12 of 89
samples with an MFI below 8317 (Mean MFI of C4d Negative + 1SD) had fixed
complement, whilst 17 of 56 samples with MFI >8317 were unable to fix
complement, suggesting the association between MFI and C4d is inconsistent.
Furthermore, analysis of patients who had undergone transplant with donor specific
Luminex detected antibodies revealed that C4d fixing antibodies were a better
predictor of poor graft survival than patients with high MFI DSA. Patients who were
C4d positive had a 90 day and 1 year graft survival of 30% and 20% respectively
compared to 58% and 42% for patients with high MFI (defined as MFI>8317). These
results suggest an inconsistent association between MFI and ability to fix C4d; they
also suggest that C4d fixation is a better predictor of poor graft survival than MFI of
Imunoglobulin subtypes: In humans, IgG1 and IgG3 are the IgG subtypes known to
activate complement via the classical pathway. This response is triggered when C1q
binds to the C 2 region of IgG1 or IgG3 or the C 3 region of IgM. Identification of
the IgG subtypes in 50 HLA antibody positive serum samples was performed using
secondary antibodies to IgG1, IgG2, IgG3 and IgG4 in Luminex assays. PE-
conjugated secondary antibodies (diluted 1/50) were used instead of the PE-labelled
anti-human IgG, which is normally used. IgG1 was the predominant isotype, being
present in 36/50 of the samples; it was the sole IgG isotype in 18 cases. IgG2 was
present in 15 samples of which only 4 contained IgG2 alone. IgG3 was detected
alone in 7 samples, with IgG3 also present in a further 13 samples in combination
with other subtypes. IgG4 was found in 10 samples although on only one occasion as
the sole subtype.
There was no correlation between IgG subclass and the ability to fix complement in
the Luminex assay, with only 19 of the 44 samples that were either IgG1 or IgG3 able
to fix C4d. Of the 18 samples that contained IgG1 alone, only 6 fixed C4d. It is
interesting to note however, in the 12 samples which were both IgG1 and IgG3, 8
fixed complement in the Luminex assay. The samples with IgG4 alone did not fix
complement. Studies from renal centres have also reported that IgG1 is the dominant
isotype in serum from sensitised patients and that mixtures of IgG1 and IgG3 are
better able to fix complement than IgG1 alone ([12,13]).
Multivariate analysis of sera containing IgG1 (n=36), IgG2 (n=15), IgG3 (n=20) and
IgG 4 (n=10) donor specific HLA antibodies, and graft survival at 1,3 and 5 years
revealed no difference in graft survival. In view of the small number of samples
studies (n=51) further studies are warranted on effects of Ig subclass and graft
survival. Further work needs to done to explore synergy between Ig subtypes and
synergy between multiple antibodies binding to the same cell, since they may help
define their clinical relevance. Currently, it appears that the C4d fixing assay, together
with determination of donor specificity, is a useful test for predicting early graft
failure after cardiac transplantation.
b) Non-HLA antibodies
It is well established that antibodies to non-HLA antigens, such as vimentin 
MICA  and angiotensin-receptor antibodies are associated with rejection after
solid organ transplantation. Use of solid phase assays for determining HLA antibodies,
has resulted in a reduced incidence of accelerated acute rejection after renal
transplantation , although when it occurs the causes remain unknown. Recent data
from our laboratory has re-evaluated the role of complement fixing IgM non-HLA
antibodies in pre-transplant serum as a risk factor for early graft failure after heart
Complement fixing IgM non-HLA antibodies
One of the advantages of performing a cytotoxic cross-match with donor leukocytes is
that presence of autoreactive cytotoxic antibodies can be discovered. These antibodies
are revealed as an IgM positive cytotoxic cross-match with donor leukocytes, which
are also positive against a panel of HLA typed leukocytes, but with no particular
specificity. Some centres will check the sera against patients’ own cells, and if
positive, these are called cytotoxic autoreactive non-HLA IgM antibodies. The
majority of renal centres have reported that this antibody is not damaging to renal
allografts [19,20]. A single study of heart transplant recipients reported that appositive
IgM cross-match had no effect on graft survival  but whether these were non-
HLA abs was not determined. We performed a large retrospective study of pre-
transplant sera from 616 adult heart transplant recipients transplanted between 1991
and 2003 . All sera were tested with Luminex beads to assess presence of HLA
antibodies. Of these 616 patients, 488 were Ab negative, 59 were IgM non-HLA +ve,
58 were IgG HLA Ab +ve, 11 were IgM HLA Ab +ve. Acturial graft survival
demonstrated that survival of grafts in patients transplanted with IgM non-HLA
antibodies (n=59) was 55.9, 54.2, 49.9, and 43.4% at years one, two five and ten,
compared to 75.8, 73.7, 66.6 and 52.8% respectively for patients who were antibody
negative (Fig 3, p=0.0085). Indeed, it can be seen from Fig 3, that survival is no
worse for patients with non-HLA IgM antibody than for those with donor specific IgG
HLA antibodies (p=0.194). Multivariate analysis demonstrated that pre-transplant
IgM non-HLA abs remain a significant risk factor for poor survival after adjusting for
other poor prognostic indicators (HR 2.359, p=0.0002). The figure demonstrates that
the main effect of this IgM non-HLA ab occurs in the first year after transplantation.
Prior to 1999, solid phase immunoassays were not available to detect HLA
antibodies and it was possible to miss HLA specificities; in this study we had re-
analysed all IgM non-HLA antibody positive sera in this study with Luminex beads
and confirmed that none of them had HLA reactivity. The specificities of the IgM
antibody are not known. In many centres these IgM non-HLA antibodies are called
autoreactive antibodies. Of the 59 sera with IgM non-HLA antibodies 54% had
reactivity to recipient leukocytes. This is likely to be an underestimate of autoreactive
cases; some of the peripheral blood samples from recipients had been stored at 4
degrees, a procedure which, in our experience, inhibits detection of autoreactive
antibody. Some of the sera contained IgM antibodies to the autoantigen vimentin
(14% of our cases). It is likely that many of them were autoantibodies reacting to
non-polymorphic autoantigens, although some may be directed at polymorphic non-
HLA antigens found on leukocytes . In this study, MICA antibodies were not
investigated. Antibodies to MICA are not detected by the lymphocytic cell cross-
match  making it unlikely that MICA is responsible for the effects described here.
Although one might speculate that presence of autoimmune antibodies prior to
transplantation may be associated with pre-transplant disease, such as dilated
cardiomyopathy which is associated with autoimmunity , in this study this was
not found to be the case.
The histological criteria of antibody-mediated rejection (AMR), published in 2005 
include endothelial swelling, presence of intravascular macrophages as well as
capillary C4d positivity, with the presence of interstitial haemorrhage, acute
inflammation, vasculitis and myocyte necrosis in the later stages. Biopsies or post-
mortem specimens from 17 patients with IgM non-HLA antibodies and 7 patients
with IgG HLA were re-reviewed for evidence of AMR, using these criteria.
Retrospective staining for C4d on all available material from IgM non-HLA and IgG
positive patients demonstrated that IgM non-HLA antibodies are associated with
complement fixation in the allograft; 5/17 specimens had strong capillary staining.
Others have also shown that non-HLA IgM antibodies are associated with C4d
deposition in allografts, thus 75% of renal allografts from ABO incompatible
recipients demonstrate C4d peritubular capillary deposits A detailed review of the
histology, cardiac pathology and consensus cause of death (COD) failed to
demonstrate that any of the 17 patients with IgM non-HLA could be said to have died
of AMR, but the histology of 3 of 7 patients with IgG HLA was consistent with the
2005 criteria for AMR. All three IgG HLA patients had DSA and 2 of them were
positive for C4d on their post-mortem (PM) specimens. The detailed histological
analysis of PM or ‘last biopsies’ revealed an association between IgM non-HLA
antibodies and presence of ischaemic damage in the heart, shown in 8/17 cases. There
was also an association between IgM non-HLA antibodies and Primary Graft Failure.
There is emerging evidence for a role for the classical pathway of complement
fixation and IgM autoantibodies in the pathology of ischaemic reperfusion injury
[24,25]. For example, immunodeficient RAG-/- mice are resistant to myocardial and
intestinal re-perfusion injury, but this is restored by infusion of either natural IgM
antibodies [25,26] or IgM autoantibodies to phospholipids [27,28]or ribonuclear-
proteins. It has been suggested that IgM natural antibodies and IgM
autoantibodies bind to neo-antigens exposed on the surface of cells exposed to
ischaemia . It is likely that the antibodies in our study are low avidity
polyspecific antibodies because they work better in the cold than at room temperature
(Smith, JD, unpublished, 2008). The latter property suggests they may have
specificity for carbohydrate antigens . The fact that they work better in the cold
suggests their pathogenecity may be unique to cardiac transplantation, where patients
are subjected to moderate hypothermia during cardiopulmonary bypass, during the
transplant procedure, and the transplanted heart is subjected to cold storage. These
antibodies are very effective at fixing complement. We suggest that the presence of
IgM autoantibodies in patients at the time of implantation may result in binding of
IgM to de-novo expressed autoantigens exposed on the allograft during ischaemia. It
has been shown that IgM co-localises with complement in infarcted human
myocardium, indicating the presence of autoantigens in damaged cardiomycoytes .
Other possible targets would be apoptotic circulating cells including activated
neutrophils  and platelets  which express autoantigens on their surface [35,36].
Fixation of complement is a highly inflammatory process, having many down stream
effects [6,37] including xenograft  and allograft rejection  ; it is associated
with platelet aggregation and endothelial activation . PGF is the most common
cause of death in the first 30 days after heart transplantation . It is a poorly
understood entity, but is significantly associated with hypoxic injury of the donor
heart  making it likely that ischemia reperfusion injury is an important causative
In conclusion: Detection of complement fixing antibodies by the cytotoxic cross
match has served the transplant community well for forty years. Use of solid phase
assays to determine specificity of antibodies has advanced our knowledge of the range
of HLA antibodies which are produced but leaves a gap of knowledge regarding
function and pathogencity. Further research, using a combination of assays to
determine specificity and function will pave the way for better understanding of the
clinical relevance of antibodies in transplantation.
Comparison of CDC and Luminex assays (565 sera)
HLA Abs Class I Class II
CDC +ve/Luminex +ve 14 12 4
CDC +ve/Luminex -ve 0 0 0
CDC -ve/Luminex +ve 53 42 21
Fig 1. Comparison of detection method and donor specificity on graft survival.
Actuarial graft survival according to antibody status of pre-transplant sera: sera
contained CDC +ve Luminex + ve DSA (n=5), CDC+ve Luminex +ve non DSA
(n=4), CDC-ve/Luminex +ve DSA (n=19), CDC-ve/Luminex +ve non DSA (n=39)
and patients with no detectable HLA antibodies (n= 498). Both CDC DSA and
Luminex DSA have significantly reduced graft survival compared with other groups,
p=0.0039. Reproduced from Smith et al , with permission from Blackwell
Fig 2. Effect of C4d depositing DSA on graft survival. Actuarial graft survival
according to antibody status of pre-transplant sera; sera contained C4d+ve DSA
(n=11), C4d+ve non DSA (n=11), C4d-ve DSA (n=13), C4d-ve non DSA (n=32) and
no detectable HLA antibodies (n= 498),p=0.0002. Reproduced from Smith et al ,
with permission from Blackwell Munskaard.
Fig 3. Effect of IgM non-HLA antibodies on graft survival. Kaplan-Meier survival
for allografted hearts (n=616) according to pre-transplant antibody status. Patients
were either antibody negative (n=488), IgM non-HLA positive (n=59), IgG HLA non-
donor specific (n=36), IgG donor-specific (n=22) or IgM HLA positive (n=11).
Reproduced from Smith et al  with permission from Lippincott Williams
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Figure(s) ACCEPTED MANUSCRIPT
CDC+ve/Luminex+ve/No DSA N=4
CDC-ve/Luminex+ve/No DSA N=39
100 Negative N=498
Graft Survival %
0 100 200 300 400 500
Days Post Transplant
Fig 2 C4d -ve/No DSA N=32
C4d +ve/No DSA N=11
C4d -ve/DSA N=13
C4d +ve/DSA N=11
Graft Survival %
0 100 200 300 400 500
Days Post Transplant
IgG HLA non-donor specific
IgG HLA donor specific
Graft Survival (%)
0 500 1000 1500 2000 2500 3000
Days Post Transplant