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REVIEW ARTICLE
Luminex and antibody detection in kidney transplantation
Antonietta Picascia •Teresa Infante •
Claudio Napoli
Received: 27 January 2012 / Accepted: 6 April 2012
ÓJapanese Society of Nephrology 2012
Abstract Preformed anti-human leukocyte antigen (HLA)
antibodies have a negative effect on kidney transplantation
outcome with an increased rejection rate and reduction in
survival. Posttransplantation production of donor-specific
anti-HLA antibodies is indicative of an active immune
response and risk of transplantation rejection. For many
years the primary technique for anti-HLA antibody detec-
tion was complement-dependent cytotoxicity (CDC),
which has been integrated by solid-phase assays as HLA
antigen-coated bead methods (Luminex). This new tech-
nological approach has allowed identification of anti-HLA
antibodies, not detectable using conventional CDC method,
in patients awaiting kidney transplantation. Moreover, use
of Luminex technology has enabled better definition of
acceptable or unacceptable antigens favoring transplanta-
tion in highly immunized patients. However, there are still
many unresolved issues, including the clinical relevance of
antibodies detected with this system.
Keywords Anti-HLA antibodies Luminex technology
Single-antigen assay
Introduction
For over 40 years anti-human leukocyte antigen (HLA)
donor reactivity has been recognized as a factor influencing
kidney transplantation outcome. Patel and Terasaki [1,2]
demonstrated that presence of antibodies against recipient
HLA antigens is a risk factor for hyperacute rejection with
immediate graft loss. In literature, there is profuse evidence
that preformed anti-HLA antibodies have a deleterious
effect on kidney transplantation outcome as a result of
increased rate of rejection and reduction in survival in
sensitized patients [3,4]. Recognition of donor HLA anti-
gens by recipient immune system causes an immune
response that can lead to rejection. Moreover, blood
transfusions, pregnancies, or previous transplantations
cause the development of anti-HLA antibodies as a result of
contact with foreign antigens. Since then, patients waiting
for transplantation have been tested for presence and
specificity of anti-HLA antibodies [5,6]. Posttransplanta-
tion production of donor-specific anti-HLA antibodies is
indicative of an active immune response, and therefore
increased risk of rejection [7,8]. Indeed, several studies
have correlated presence of posttransplantation antibodies
with rejection and graft loss [8,9]. Pre transplantation,
detection and analysis of anti-HLA antibodies in patient
sera are essential for predicting the likelihood of finding a
compatible donor crossmatch, to avoid transplantation from
donor presenting HLA antigens to which the patient is
sensitized. In addition, precise identification of antibody
specificities is used to select the optimal compatibility test
to avoid a false positive, excluding clinically irrelevant
A. Picascia (&)C. Napoli
U.O.C. Immunohematology, Transfusion Medicine and
Transplant Immunology (SIMT), Regional Reference Laboratory
of Transplant Immunology (LIT), Azienda Universitaria
Policlinico (AOU), Second University of Naples,
Piazza Miraglia, 1, 80138 Naples, Italy
e-mail: antonietta.picascia@policliniconapoli.it
T. Infante C. Napoli
Fondazione SDN, IRCCS, Via Gianturco, 80134 Naples, Italy
C. Napoli
Department of General Pathology, Chair of Clinical Pathology,
and Excellence Research Centre on Cardiovascular Diseases,
Azienda Universitaria Policlinico (AOU), Second University
of Naples, Piazza Miraglia 2, 80138 Naples, Italy
123
Clin Exp Nephrol
DOI 10.1007/s10157-012-0635-1
antibodies, and to identify patients at high risk of organ
rejection [4].
The complement-dependent cytotoxicity (CDC) assay
has been for many years the gold-standard technique for
anti-HLA antibody detection [2]. In recent years new solid-
phase techniques, such as enzyme-linked immunosorbent
assay (ELISA)-based and HLA antigen-coated bead (Lum-
inex) methods, have been introduced, allowing better
understanding of the role of anti-HLA antibodies in organ
rejection [10–13]. The development of more sensitive and
specific solid-phase approaches has completely revolution-
ized detection of anti-HLA antibodies. However, the clinical
relevance of anti-HLA antibodies identified with these
techniques remains to be understood, despite the achieve-
ment of universal consensus [14,15]. Today, the most used
method for anti-HLA antibody detection is based on fluo-
rescent beads with Luminex technology, but many technical
and interpretive issues remain unclear. In this review, we
analyze advantages and issues regarding use of Luminex in
anti-HLA antibody detection in kidney transplantation.
Classical methods for anti-HLA antibody detection
Several techniques are currently used for detection of anti-
HLA antibodies to decrease the rate of organ rejection and
improve survival: CDC, ELISA, and bead-based assays
(Luminex and FlowPRA).
Complement-dependent cytotoxicity
Complement-dependent cytotoxicity is a ‘‘cell-based’’
assay in which antibodies, present in patient sera, bind to
HLA antigens expressed on the lymphocyte membrane. The
serum is incubated with cells from a HLA-typed panel to
allow formation of an immune complex which, after com-
plement addition, results in cell lysis [1]. Lysed cells are
stained by a fluorescent dye to discriminate positive and
negative reactions. The result is expressed as a panel reac-
tive antibody (PRA) value, defined as the percentage of
cells in the panel that give a positive reaction with serum.
Using CDC assay, detection and assignment of class I anti-
HLA antibodies is less difficult for sensitized patients with
low PRA than for highly immunized patients. Furthermore,
class II anti-HLA antibody detection is complicated by the
presence of both class I and II molecules on B lymphocyte
membrane and by low rate of these cells in peripheral blood.
In addition, the CDC identifies complement-activating IgG
and IgM antibodies. Most anti-HLA antibodies are IgG,
while IgM are frequently autoantibodies whose clinical
role is still debated. To solve this issue, serum pretreatment
with dithiothreitol (DDT) is used for elimination of these
antibodies [16]. Moreover, CDC has several technical
problems including the need for a large panel of live cells
expressing the most common HLA antigens, the presence of
autoantibodies, and the difficulty in distinguishing between
class I and II anti-HLA antibodies. Furthermore, several
cytolytic pre- and posttransplantation therapies may inter-
fere with cell assay [17]. Nevertheless, CDC has the
advantage of reflecting the situation in vivo, because HLA
antigens, used as antibody target, are in their native con-
figuration on cell membrane. Over the years, changes have
been introduced to increase CDC sensitivity, such as anti-
human globulin (AHG) addition and prolonged incubation
times [18]. Therefore, the CDC reactivity pattern is a useful
indicator of increased risk of hyperacute or acute rejection,
and a positive CDC test is still considered a contraindication
to transplantation in many centers.
Enzyme-linked immunosorbent assay
Solid-phase or ‘‘membrane-independent’’ assays include
ELISA and flow technology. In these methods, purified
HLA antigens are coated onto wells of a plate or onto beads.
The ELISA test uses recombinant or soluble HLA mole-
cules, immunoprecipitates from platelets or Epstein–Barr
virus, that are directly bound to wells of microplates. Anti-
HLA antibodies, if present in patient serum, bind to the
HLA antigen. Subsequently, a secondary enzyme-conju-
gated antibody IgG is added, followed by a substrate addi-
tion that induces a colorimetric reaction indicating antibody
reactivity. Two types of test are available: The first uses
plates with a large number of different class I and II HLA
antigens and provides only a positive or negative result for
anti-HLA antibody presence. A second assay, instead using
plates with HLA molecules of one individual bound to
wells, provides identification of antibody specificity. ELISA
is a more sensitive approach than the CDC method. It is a
semiquantitative assay that detects both complement-acti-
vating and non-complement-activating antibodies [11,12].
Bead-based assays (Luminex technology)
Luminex technology is a high-throughput platform for anti-
HLA-specific antibody detection consisting in a flow-based
bead assay [13,19,20] (Fig. 1). Antibody screening is per-
formed with a set of polystyrene microspheres containing
different fluorochromes, coated with a specific or different
HLA molecules. The recipient serum is added to the bead
mix, and anti-HLA antibodies, if present, bind to specific
antigen coated on microspheres. A second phycoerythrin-
labeled anti-human IgG antibody (PE) is then added, which
binds to the primary anti-HLA antibody. All bead mix con-
tains a positive control bead coated with IgG and a negative
control bead without HLA molecules. A negative control
serum is run in every assay to establish the background for
Clin Exp Nephrol
123
each bead. The Luminex system is used for detection,
acquisition, and data analysis. The Luminex analyzer is a
flow cytometer with an excitation system comprising two
solid-state lasers. The red classification laser excites the
fluorochromes in beads, while the green reporter laser excites
the fluorescence of the PE molecules bound to anti-HLA
antibodies on each bead. The combination of the two signals
defines antibody specificity (Fig. 1a). The fluorescence sig-
nal of the PE label is expressed as a median fluorescence
intensity (MFI) bead value. Serum reactivity can be evalu-
ated by the fluorescence signal for each bead after correction
for nonspecific binding to the negative control bead. All data
are normalized with the negative control serum. There are
three levels of analysis: mixed or screening, PRA or identi-
fication, and single-antigen (SA) assays. The first level
consists in serum screening, with beads bound with a wide
number of purified class I and II HLA antigens, essentially
providing a positive or negative result. The PRA level
determines anti-HLA antibody specificity using beads
coated with the phenotype equivalent of one cell (Fig. 1b).
Regarding SA, beads are coated with single recombinant
antigens from transfected human cell lines, allowing accu-
rate definition of anti-HLA antibody specificities (Fig. 1c).
Bead-based assays (FlowPRA)
FlowPRA is a flow cytometry technique used for detection
of both HLA class I and II antibodies. There are three
different levels of analysis with these FlowPRA tests:
FlowPRA screening, specific, and FlowPRA single anti-
gen. The FlowPRA screening test is preformed with two
pools of microbeads coated with different HLA class I and
II antigens purified by affinity. Common and rare HLA
antigens are represented within these pools. Class I and II
beads have different fluorescence properties. FlowPRA
specific class I and class II tests consist in a panel of
different beads coated with different purified class I or
class II antigens. FlowPRA class I or class II single-anti-
gen tests, instead, consist of beads coated with single HLA
antigens, produced by recombinant technology. The pro-
cedure is the same for different FlowPRA tests. Each
group of beads is incubated separately with the patient
sera and stained with fluorescein isothiocyanate (FITC)-
conjugated anti-human IgG. Both acquisition and analysis
are performed by a flow cytometer in which beads can be
excited at 488 nm, generating a maximum emission of
approximately 580 nm, detected by the FL2 channel.
Since different beads generate different FL2 channel
shifts, different colored beads in a group can be separated
by a flow cytometer on the FL2 channel. Beads reacting
positively show a FL1 channel shift on FL1 versus FL2
dot plots compared with serum negative control and con-
trol beads, allowing determination of HLA specificity.
Positive and negative control sera are used to set the cutoff
line on the FL1 versus FL2 dot plot for each bead group
[21,22].
Fig. 1 Basic principles of Luminex technology. aFluorescent beads
coated with HLA antigens bind antibodies present in serum. The red
laser excites the fluorochrome within the beads, while the green laser
detects the fluorescence signal of PE conjugated to the secondary
antibody. Data are acquired, processed, and analyzed by the Luminex
platform. The fluorescence intensity, expressed as MFI, is represented
by bar graphs.bExample PRA-level analysis, in which beads are
coated with the equivalent phenotype of one cell. cExample single-
antigen bead analysis, where each bead is coated with a specific HLA
antigen
Clin Exp Nephrol
123
The FlowPRA assay is comparable to the Luminex
assay in the identification and characterization of HLA
antibodies [19].
Recent advantages in anti-HLA antibody detection
by Luminex
Luminex single-antigen assay
The major advantage of Luminex technology in anti-HLA
antibody detection, especially using SA analysis, is the
accurate evaluation of complex sera from highly immu-
nized patients for whom it is very difficult to identify
antibody specificity and, therefore, find a compatible donor
[23,24]. Each anti-HLA antibody may bind multiple
epitopes, structurally defined by few amino-acid residues
common to several class I and II HLA molecules [25]. The
Luminex SA assay provides precise distinction between
class I and II anti-HLA antibodies. Indeed it is possible to
discriminate clearly different antibodies directed against
class II molecules, as HLA-DRB1,3,4,5, HLA-DQB1,
HLA-DQA1, and HLA-DPB [26,27].
However, the recent development of microspheres
coated with HLA-DP, HLA-C, and HLA-DQA antigens
has allowed exploration of the role of these antibodies in
kidney transplantation [28–30]. Vaidya et al. [31] reported
the case of a patient in end-stage renal disease, who pre-
sented a positive crossmatch with B cells with a donor to 0
mismatch on loci HLA-A, HLA-B, HLA-C, HLA-DRB1,
and HLA-DQB1. The positive crossmatch was the result of
a mismatch on the HLA-DPB locus. Several studies have
shown that mainly anti-HLA-C and anti-HLA-DP donor-
specific antibodies were found in patients displaying acute
rejection [28–30]. Pretransplantation anti-HLA-DP anti-
bodies seemed to be involved more frequently in poor graft
outcome, as shown in several recently published cases.
Thaunat et al. [32] highlighted the case of a kidney-trans-
planted patient in whom detection of anti-HLA-DP anti-
bodies preceded chronic rejection onset. Interestingly, the
patient had, in addition to anti-HLA-DP donor-specific
antibodies, even anti-HLA-DP non-donor-specific anti-
bodies. Pre- and posttransplantation sera of 338 patients
were screened for anti-HLA-DP antibodies using the
Luminex SA assay. Pretransplantation anti-HLA-DP anti-
bodies were detected in 23 % of patients, and posttrans-
plantation anti-HLA-DP antibody development was found
in 77 % of them. This study showed that anti-HLA-DP
antibodies are specific for epitopes shared by different
HLA-DP antigens, suggesting that matching for immuno-
genic HLA-DP epitopes in kidney transplantation appears
to be more relevant than classical allele matching to pre-
vent HLA-DP immunization [33].
The involvement of anti-HLA-C, anti-HLA-DQA, anti-
HLA-DQB, and anti-HLA-DP antibodies in organ rejec-
tion, detected through SA assay, could revolutionize
kidney allocation criteria [34–36].
Another great advantage of SA is the ability to detect
antibodies against specific HLA alleles. In kidney trans-
plantation, several studies have demonstrated the presence
of allele-specific antibodies [37,38]. Proust et al. [37]
reported donor-specific antibodies against specific HLA-
DR and HLA-DQ alleles in a nonimmunized patient
transplanted with a kidney that was HLA-DR and DQ
identical. This study emphasizes that minimizing HLA-DR
and HLA-DQ mismatches is not sufficient to prevent
alloimmunization, and therefore identification of epitope or
antigenic determinant binding antibodies is of paramount
importance. Indeed, high HLA-A, HLA-B, and HLA-DR
compatibility does not always guarantee successful kidney
transplantation, suggesting the involvement of other anti-
gens in organ rejection.
MICA antibodies
Recent studies have shown that anti-major histocompati-
bility complex class I-related chain A (MICA) antibodies,
using MICA molecules bound to Luminex beads, have
important clinical significance in transplantation. Pre-
transplantation detection of anti-MICA antibodies is asso-
ciated with increased risk of rejection and lower graft
survival. Furthermore, posttransplantation anti-MICA
antibody development is related to greater rate of rejection
and graft failure. Presence of anti-MICA antibodies could
be an important diagnostic marker of rejection, suggesting
a pivotal role for their identification and monitoring
[39,40].
Virtual and Luminex crossmatch
The ability of Luminex to identify anti-HLA antibodies has
improved the definition of acceptable and unacceptable
antigens, facilitating exchange of organs between different
centers to facilitate transplantation in highly immunized
patients [41]. Nevertheless, several studies have shown that
low antibody levels are not always associated with adverse
effects. Therefore, clinically irrelevant anti-HLA antibody
detection could lead to discrimination against some
immunized patients at time of organ allocation, if clinically
irrelevant antibodies are evaluated as assigning an unac-
ceptable antigen in virtual compatibility crossmatch
(VXM) [42]. VXM allows exclusion of donors who express
HLA antigens against which a patient is immunized.
Zachary et al. demonstrated that accurate identification and
determination of donor-specific antibody strength, detected
by solid-phase approaches, correlates with CDC and flow
Clin Exp Nephrol
123
cytometry crossmatches. This correlation allowed predic-
tion of the result of flow cytometry and CDC crossmatches
in 92.8 and 92.4 % of cases. One of the major issues in
virtual crossmatch using solid-phase assays is nonstan-
dardized interpretation; therefore, antibody strength and
titer, complement fixation, and sensitization events must be
accurately considered [41,43,44]. Gandhi et al. [45], in a
retrospective study on heart transplantation, defined a vir-
tual crossmatch as negative when MFI values, referring to
antibodies identified by SA, were \300, and as positive for
MFI values [1500.
Recently, a new system exploiting Luminex technology
has been developed to perform a prospective crossmatch.
The Luminex crossmatch (LXM) is performed using
microspheres coated with antibodies directed against
class I and II HLA antigens, which specifically bind to
donor HLA molecules. Subsequently, patient serum is
incubated with beads coated with donor HLA antigens.
After addition of a IgG-phycoerythrin conjugated second-
ary antibody, fluorescence is read using the Luminex
instrument. Billen et al. [46] reported, for the first time, a
study on donor-specific Luminex crossmatch. Results
obtained were compared with those of flow cytometry
crossmatch, performed on the same donor–recipient pairs.
The sensitivity and specificity of the LXM were 89 and
98 % for class I and 68 and 97 % for class II, respectively.
Subsequently, sera were tested with SA to define donor-
specific anti-HLA antibodies. The increased sensitivity for
class I rather than class II could be due to nondetection of
all anti-HLA-DQ antibodies, or insufficient detection of
anti-HLA-DP; only anti-HLA-DR antibodies appear to be
correctly identified. In a recent study, results of SA anti-
body detection have been compared with the LXM using
sera already studied by CDC. Data reported have shown
good correlation between LXM and SA for class I and for
HLA-DR antibodies [47]. In general, sensitivity was higher
using SA compared with LXM for both anti-class I and II
antibodies, probably as each bead is coated with a single
antigen. These studies have further confirmed that LXM
does not detect anti-HLA-DQ antibodies [48]. Recently,
another study evaluated clinical detection of LXM in a
group of recipients with pretransplantation anti-HLA anti-
bodies. That report showed a correlation between class I
LXM and antibody-mediated acute rejection (AMR),
combining crossmatch results with those of SA. Episodes
of AMR were shown in 80 % of patients positive for class I
LXM and with MFI values [900 in single antigen [49].
Class I LXM results are more sensitive than flow cytom-
etry crossmatch, so it could be a useful tool to assess HLA
compatibility in organ transplantation [49]. Unlike cell-
based CDC and flow cytometry crossmatch assays, LXM
does not require living cells. In addition, cell lysates can be
stored and used to monitor the possible production of
donor-specific antibodies or the effectiveness of immuno-
suppressive therapy.
Impact of anti-HLA antibodies on clinical outcome
It is well known that solid-phase technology is more sen-
sitive and specific than CDC to detect anti-HLA antibodies,
and the Luminex approach is certainly more sensitive than
ELISA [13,17,50,51]. However, the clinical relevance of
antibodies detected by high-sensitivity assays is unclear
and remains a debated topic [23,52]. In this regard, several
retrospective studies have evaluated the clinical signifi-
cance of anti-HLA antibodies detected by Luminex in
recipients with negative pretransplantation CDC, showing
different results. Gupta et al. [53] tested sera of renal
transplantation patients by Luminex; 13 % of patients
showed donor-specific anti-HLA antibodies, undetectable
by cytotoxicity at time of transplantation, and 18 % of
patients with non-donor-specific anti-HLA antibodies.
There were no cases of hyperacute rejection in either
group. These data emphasize that a negative CDC cross-
match, in the presence of donor-specific anti-HLA anti-
bodies at time of transplantation, has little impact on all
early graft parameters but these antibodies are associated
with poorer long-term outcome [54].
In contrast, another study by van den Berg-Loonen [23]
showed that highly sensitized patients, transplanted with a
negative CDC crossmatch and with donor-specific anti-
HLA antibodies, detectable only by Luminex, presented
excellent long-term graft survival.
In clinical practice, more sensitive approaches in anti-
HLA antibody detection have allowed it to be demonstrated
that donor-specific anti-HLA antibody preexistence is not
always a contraindication to transplantation. Nevertheless,
the main issue is to establish a consensus on the parameters
that predict clinical relevance of donor-specific anti-HLA
antibodies, in order to discriminate between clinically and
non-clinically relevant antibodies [53]. For this purpose, the
definition of antibody specificities in patient serum is crucial
[55,56]. Today, knowledge of epitopes on HLA antigens has
facilitated identification of antibody specificity; however,
not all antibodies are clinically relevant. Indeed, solid-phase
assays employ purified antigens which may have a different
conformation from HLA molecules expressed on cell
membrane [57–59]. In light of this, a positive CDC cross-
match, due to specific donor anti-HLA antibodies, still has
important clinical significance. In this regard, expression of
target molecules on the graft and antibody titer should be
evaluated. However, the high level of sensitivity in antibody
detection does not necessarily reflect their clinical relevance.
Patients positive only on Luminex assay have low HLA
antibody titer compared with patients positive also on CDC.
Clin Exp Nephrol
123
The sensitivity of SA depends on the cutoff value used
to discriminate between positive and negative reactions and
to predict donor-specific CDC crossmatch [43,60,61]; this
cutoff point may vary considerably among laboratories.
Many cutoff points have been described in literature, with a
median range of MFI ranging from 500 to 6000 [42,45,56,
62]. To reduce analytical variability and compare results
between different laboratories, another unit of fluorescence
has been introduced, defined as molecules of equivalent
soluble fluorochromes (MESF). In particular, Mizutani
et al. [62] showed that antibody titer is directly correlated
to MESF values, with a strong association between donor-
specific MESF and graft failure. Vaidya et al. [63]
measured concentrations of anti-HLA antibodies in multi-
specific sera by converting fluorescence intensity into
MESF units and showing that antibodies detected by CDC
have a threshold value corresponding to MESF of 250000,
below which the CDC assay is negative. These studies have
shown that antibodies detected by Luminex with lower
values of MESF may not have deleterious effects on short-
time outcome, while long-term effects remain to be
clarified.
Complement- and non-complement-activating
antibodies in transplantation
Unlike the CDC technique, Luminex technology, as well as
all solid-phase approaches that do not rely on complement
fixation, are not able to distinguish between complement-
activating and non-complement-activating antibodies.
Today, the strong and deleterious impact of complement-
fixing antibodies on rejection and graft failure has been
widely demonstrated. However, the clinical significance of
nonactivating complement antibodies and their involvement
in organ rejection is still debated, so it could be useful to
know the nature of antibodies involved in immune response
[20,50]. Several modifications were made to solid-phase
assays to detect only complement-fixing antibodies [64,65].
Wahrmann et al. [65] developed a novel cell-independent
assay for assessment of complement-activating panel reac-
tivity using human serum as source of C4d complement,
which binds to beads in presence of complement-binding
HLA antibodies. Authors have shown, with this approach, a
highly significant association between absolute C4d-Flow-
PRA and CDC-PRA levels. Smith et al. [66] changed the
previous approach adapted to the Luminex SA, using
different secondary antibodies for complement-activating
anti-HLA antibody detection. Data showed an effect of non-
complement-activating antibodies on graft survival, com-
pared with the patient with absence of anti-HLA antibodies.
Heinemann et al. [67] replaced the total IgG with individual
isotypes, such as IgG1, 2, 3, or 4, showing that non-
complement-activating anti-HLA antibodies accumulate in
rejected kidney grafts, even if their effect is not clear. Other
studies have shown high incidence of non-complement-
activating anti-HLA antibody presence in patients on
transplantation waiting list [68,69]. Recently, a new assay
has been developed to detect immunoglobulin subclasses
fixing complement, through C1q identification on the
Luminex platform [70], although the role of these antibodies
in both rejection and graft failure is unclear. More investi-
gations are required to discriminate between activating and
non-complement-activating antibodies [65,71]. Chen et al.
[72] suggested that complement-fixing antibodies, regard-
less of the strength of IgG MFI, represent a component that
may influence clinical outcome.
Conclusions
In clinical practice, the introduction of new and more
sensitive approaches for detection of anti-HLA-antibodies,
such as the fluorescent antigen-coated bead assay, has
allowed identification of HLA immunization in patients
considered not immunized on CDC. Furthermore, these
assays have enabled disclosure of antibodies against other
loci such as HLA-C, HLA-DP, HLA-DQ, and MICA and
allele-specific antibodies in transplantation recipients. If
further studies prove the clinical impact of these antibod-
ies, criteria for kidney allocation could be revolutionized.
Technological improvements have revealed antibodies
against epitopes shared by multiple HLA antigens. Better
understanding of all HLA epitopes could provide a more
efficient approach to determine histocompatibility and
influence organ allocation, suggesting that matching for
immunogenic epitopes appears to be more relevant than
classical allele matching to prevent immunization.
The clinical relevance of anti-HLA antibodies identified
with these systems remains a debated issue. Indeed, it
should be emphasized that, in solid-phase assays, HLA
molecules are manipulated and their natural conformation
might be altered with exposure or loss of antigenic epitopes
determining false-positive or false-negative results. In
addition, in vivo antigen density on beads does not always
reflect natural expression of HLA molecules on cells.
These methods might increase the likelihood of trans-
plantation in immunized patients through the definition of
acceptable HLA mismatches, but detection of clinically
irrelevant anti-HLA antibodies could lead to discrimination
against sensitized patients at organ allocation. An unre-
solved issue is the lack of universal consensus on the
definition of cutoff values for donor-specific antibody
strength to predict a positive crossmatch.
Moreover, these assays can be used also to monitor the
development of clinically relevant anti-HLA antibodies
after transplantation, facilitating better management of
Clin Exp Nephrol
123
patients. Despite these technical improvements, results
obtained are affected by many unresolved issues, including
the clinical impact of complement-activating and non-
complement-activating antibodies and the role of non-
anti-HLA antibodies in graft failure. Therefore, accurate
analysis of sensitizing events pre transplantation is
important, and integration of multiple methods must be
taken into account to characterize the immunologic status
of patients awaiting transplantation.
Acknowledgments We thank Dr. Vincenzo Grimaldi for his
support.
Conflict of interest None.
References
1. Patel R, Terasaki PI. Significance of the positive crossmatch test
in kidney transplantation. New Engl J Med. 1969;280:735–9.
2. Terasaki PI. Humoral theory of transplantation. Am J Transpl.
2003;3:665–73.
3. Lefaucheur C, Loupy A, Hill GS, Andrade J, Nochy D, Antoine
C, et al. Preexisting donor-specific HLA antibodies predict out-
come in kidney transplantation. J Am Soc Nephrol. 2010;218:
1398–406.
4. Kaneku H. Impact of donor-specific HLA antibodies in trans-
plantation, a review of the literature published in the last three
years. Clin Transpl. 2010;283–306.
5. Loupy A, Suberbielle-Boissel C, Hill GS, Lefaucheur C, Angli-
cheau D, Zuber J, et al. Outcome of subclinical antibody-medi-
ated rejection in kidney transplant recipients with preformed
donor-specific antibodies. Am J Transpl. 2009;9:2561–70.
6. Lachmann N, Terasaki PI, Budde K, Liefeldt L, Kahl A, Reinke
P, et al. Anti-human leukocyte antigen and donor-specific anti-
bodies detected by luminex posttransplant serve as biomarkers for
chronic rejection of renal allografts. Transplantation. 2009;87:
1505–13.
7. Lee PC, Zhu L, Terasaki PI, Everly MJ. HLA-specific antibodies
developed in the first year posttransplant are predictive of chronic
rejection and renal graft loss. Transplantation. 2009;88:568–74.
8. Zhu L, Lee PC, Everly MJ, Terasaki PI. Detailed examination of
HLA antibody development on renal allograft failure and func-
tion. Clin Transpl. 2008;171–87.
9. Gloor JM. The utility of comprehensive assessment of donor
specific anti-HLA antibodies in the clinical management of
pediatric kidney transplant recipients. Pediatr Transpl. 2011;15:
557–63.
10. Kao KJ, Scornik JC, Small SJ. Enzyme-linked immunoassay for
anti-HLA antibodies—an alternative to panel studies by lym-
phocytotoxicity. Transplantation. 1993;55:192–6.
11. Monien S, Salama A, Scho
¨nemann C. ELISA methods detect
HLA antibodies with variable sensitivity. Int J Immunogenet.
2006;33:163–6.
12. Pajaro OE, George JF. On solid-phase antibody assays. J Heart
Lung Transpl. 2010;29:1207–9.
13. Colombo MB, Haworth SE, Poli F, Nocco A, Puglisi G, Inno-
cente A, et al. Luminex technology for anti-HLA antibody
screening: evaluation of performance and of impact on laboratory
routine. Cytometry B ClinCytom. 2007;72:465–71.
14. Ozawa M, Verbanac KM, Catrou P, Haisch CE, Terasaki PI.
Clinical and anti-HLA antibody profile of nine renal transplant
recipients with failed grafts: donor-specific and non-donor-spe-
cific antibody development. Clin Transpl. 2006;241–53.
15. El-Awar N, Lee J, Terasaki PI. HLA antibody identification with
single antigen beads compared to conventional methods. Hum
Immunol. 2005;66:989–97.
16. Arnold ML, Zacher T, Dechant M, Kalden JR, Doxiadis II, Sprie-
wald BM. Detection and specification of noncomplement binding
anti-HLA alloantibodies. Hum Immunol. 2004;65:1288–96.
17. Zeevi A, Girnita A, Duquesnoy R. HLA antibody analysis:
sensitivity, specificity, and clinical significance in solid organ
transplantation. Immunol Res. 2006;36:255–64.
18. Zachary AA, Klingman L, Thorne N, Smerglia AR, Teresi GA.
Variations of the lymphocytotoxicity test. An evaluation of
sensitivity and specificity. Transplantation. 1995;60:498–503.
19. Tait BD. Solid phase assays for HLA antibody detection in
clinical transplantation. Curr Opin Immunol. 2009;21:573–7.
20. Tait BD, Hudson F, Cantwell L, Brewin G, Holdsworth R,
Bennett G, et al. Review article: luminex technology for HLA
antibody detection in organ transplantation. Nephrology (Carlton).
2009;14:247–54.
21. Pei R, Wang G, Tarsitani C, Rojo S, Chen T, Takemura S, et al.
Simultaneous HLA Class I and Class II antibodies screening with
flow cytometry. Hum Immunol. 1998;59:313–22.
22. Pei R, Lee JH, Shih NJ, Chen M, Terasaki PI. Single human
leukocyte antigen flow cytometry beads for accurate identifica-
tion of human leukocyte antigen antibody specificities. Trans-
plantation. 2003;75:43–9.
23. Van den Berg-Loonen EM, Billen EV, Voorter CE, van Heurn
LW, Claas FH, van Hooff JP, et al. Clinical relevance of pre-
transplant donor-directed antibodies detected by single antigen
beads in highly sensitized renal transplant patients. Transplanta-
tion. 2008;85:1086–90.
24. Cecka JM. Current methodologies for detecting sensitization to
HLA antigens. Curr Opin Organ Transpl. 2011;16:398–403.
25. Cai J, Terasaki PI. Post-transplantation antibody monitoring and
HLA antibody epitope identification. Curr Opin Immunol. 2008;
20:602–6.
26. El-Awar N, Terasaki PI, Cai J, Deng CT, Ozawa M, Nguyen A.
Epitopes of the HLA-A, B, C, DR, DQ and MICA antigens. Clin
Transpl. 2009;295–321.
27. Deng CT, El-Awar N, Ozawa M, Cai J, Lachmann N, Terasaki
PI. Human leukocyte antigen class II DQ alpha and beta epitopes
identified from sera of kidney allograft recipients. Transplanta-
tion. 2008;86:452–9.
28. Qiu J, Cai J, Terasaki PI, El-Awar N, Lee JH. Detection of
antibodies to HLA-DP in renal transplant recipients using single
antigen beads. Transplantation. 2005;80:1511–3.
29. Singh P, Colombe BW, Francos GC, Martinez Cantarin MP,
Frank AM. Acute humoral rejection in a zero mismatch deceased
donor renal transplant due to an antibody to an HLA-DP alpha.
Transplantation. 2010;90:220–1.
30. Gilbert M, Paul S, Perrat G, Giannoli C. Pouteil Noble C,
Morelon E et al. Impact of pretransplant human leukocyte anti-
gen-C and -DP antibodies on kidney graft outcome. Transpl Proc.
2011;43:3412–4.
31. Vaidya S, Hilson B, Sheldon S, Cano P, Fernandez-Vina M. DP
reactive antibody in a zero mismatch renal transplant pair. Hum
Immunol. 2007;68:947–9.
32. Thaunat O, Hanf W, Dubois V, McGregor B, Perrat G, Chauvet
C, et al. Chronic humoral rejection mediated by anti-HLA-DP
alloantibodies: insights into the role of epitope sharing in donor-
specific and non-donor specific alloantibodies generation. Transpl
Immunol. 2009;20:209–11.
33. Billen EV, Christiaans MH, Doxiadis II, Voorter CE, van den
Berg-Loonen EM. HLA-DP antibodies before and after renal
transplantation. Tissue Antigens. 2010;75:278–85.
Clin Exp Nephrol
123
34. Duquesnoy RJ, Marrari M. Detection of antibodies against
HLA-C epitopes in patients with rejected kidney transplants.
Transpl Immunol. 2011;24:164–71.
35. Barabanova Y, Ramon DS, Tambur AR. Antibodies against
HLA-DQ alpha-chain and their role in organ transplantation.
Hum Immunol. 2009;70:410–2.
36. Bryan CF, Luger AM, Smith JL, Warady BA, Wakefield M,
Schadde E, et al. Sharing kidneys across donor-service area
boundaries with sensitized candidates can be influenced by HLA
C. Clin Transpl. 2010;24:56–61.
37. Proust B, Kennel A, Ladrie
`re M, Kessler M, Perrier P. Unex-
pected anti-HLA-DR and -DQ alloantibodies after nephrectomy
of an HLA-DR and -DQ identical first renal transplant. Transpl
Immunol. 2009;21:166–8.
38. Muro M, Gonza
´lez-Soriano MJ, Salgado G, Lo
´pez R, Boix F,
Lo
´pez M, et al. Specific ‘‘intra-allele’’ and ‘‘intra-broad antigen’’
human leukocyte antigen alloantibodies in kidney graft trans-
plantation. Hum Immunol. 2010;71:857–60.
39. Cox ST, Stephens HA, Fernando R, Karasu A, Harber M, Howie
AJ, et al. Major histocompatibility complex class I-related chain
A allele mismatching, antibodies, and rejection in renal trans-
plantation. Hum Immunol. 2011;72:827–34.
40. Narayan S, Tsai EW, Zhang Q, Wallace WD, Reed EF, Ettenger
RB. Acute rejection associated with donor-specific anti-MICA
antibody in a highly sensitized pediatric renal transplant recipient.
Pediatr Transpl. 2011;15:E1–7.
41. Zachary AA, Montgomery RA, Leffell MS. Defining unaccept-
able HLA antigens. Curr Opin Organ Transpl. 2008;13:405–10.
42. Zachary AA, Sholander JT, Houp JA, Leffell MS. Using real data
for a virtualcrossmatch. Hum Immunol. 2009;70:574–9.
43. Batal I, Zeevi A, Lunz JG 3rd, Aggarwal N, Shapiro R, Randh-
awa P, et al. Antihuman leukocyte antigen–specific antibody
strength determined by complement-dependent or solid-phase
assays can predict positive donor-specific crossmatches. Arch
Pathol Lab Med. 2010;134:1534–40.
44. Billen EV, Christiaans MH, van den Berg-Loonen EM. Clinical
relevance of Luminex donor-specific crossmatches: data from
165 renal transplants. Tissue Antigens. 2009;74:205–12.
45. Gandhi MJ, Degoey SR, Bundy K, Kremers WK, Knauer R,
Pereira N, et al. Effect of pretransplant human leukocyte antigen
antibodies detected by solid-phase assay on heart transplant
outcomes. Transplant Proc. 2011;43(10):3840–6.
46. Billen EV, Voorter CE, Christiaans MH, van den Berg-Loonen
EM. Luminex donor-specific crossmatches. Tissue Antigens.
2008;71:507–13.
47. Caro-Oleas JL, Gonza
´lez-Escribano MF, Toro-Llamas S, Acev-
edo MJ, Martinez-Bravo MJ, Aguilera I, et al. Donor-specific
antibody detection: comparison of single antigen assay and
Luminex crossmatches. Tissue Antigens. 2010;76:398–403.
48. Caro-Oleas JL, Gonza
´lez-Escribano MF, Gonza
´lez-Roncero FM,
Acevedo-Calado MJ,Cabello-Chaves V, Gentil-Govantes MA
et al. Clinical relevance of HLA donor-specific antibodies
detected by single antigen assay in kidney transplantation.
Nephrol Dial Transpl. 2011 [Epub ahead of print].
49. Riethmu
¨ller S, Ferrari-Lacraz S, Mu
¨ller MK, Raptis DA, Hadaya
K, Ru
¨si B, et al. Donor-specific antibody levels and three gen-
erations of crossmatches to predict antibody-mediated rejection
in kidney transplantation. Transplantation. 2010;90:160–7.
50. Leffell MS, Zachary AA. Antiallograft antibodies: relevance,
detection, and monitoring. Curr Opin Organ Transpl. 2010;15:
2–7.
51. Minucci PB, Grimaldi V, Casamassimi A, Cacciatore F, Som-
mese L, Picascia A, et al. Methodologies for Anti-HLA antibody
screening in patients awaiting kidney transplant: a comparative
study. Exp Clin Transpl. 2011;9(6):381–6.
52. Poli F, Cardillo M, Scalamogna M. Clinical relevance of human
leukocyte antigen antibodies in kidney transplantation from
deceased donors: the North Italy Transplant program approach.
Hum Immunol. 2009;70:631–5.
53. Gupta A, Sinnott P. Clinical relevance of pretransplant human
leukocyte antigen donor-specific antibodies in renal patients
waiting for a transplant: a risk factor. Hum Immunol. 2009;70:
618–22.
54. Gupta A, Iveson V, Varagunam M, Bodger S, Sinnott P, Thu-
raisingham RC. Pretransplant donor-specific antibodies in cyto-
toxic negative crossmatch kidney transplants: are they relevant?
Transplantation. 2008;85:1200–4.
55. Eng HS, Bennett G, Bardy P, Coghlan P, Russ GR, Coates PT.
Clinical significance of anti-HLA antibodies detected by Lum-
inex: enhancing the interpretation of CDC-BXM and important
post-transplantation monitoring tools. Hum Immunol. 2009;70:
595–9.
56. Aubert V, Venetz JP, Pantaleo G, Pascual M. Low levels of
human leukocyte antigen donor-specific antibodies detected by
solid phase assay before transplantation are frequently clinically
irrelevant. Hum Immunol. 2009;70:580–3.
57. Opelz G, Claas FH. Which human leukocyte antigen antibodies
are really clinically relevant? Hum Immunol. 2009;70:561–2.
58. Zoet YM, Brand-Schaaf SH, Roelen DL, Mulder A, Claas FH,
Doxiadis II. Challenging the golden standard in defining donor-
specific antibodies: does the solid phase assay meet the expec-
tations? Tissue Antigens. 2011;77:225–8.
59. Vlad G, Ho EK, Vasilescu ER, Colovai AI, Stokes MB,
Markowitz GS, et al. Relevance of different antibody detection
methods for the prediction of antibody-mediated rejection and
deceased-donor kidney allograft survival. Hum Immunol. 2009;
70:589–94.
60. Pereira S, Perkins S, Lee JH, Shumway W, LeFor W, Lopez-
Cepero M et al. Donor-specific antibody against denatured HLA-
A1: clinically nonsignificant? Hum Immunol. 2011;72:492–8.
61. Taylor CJ, Kosmoliaptsis V, Summers DM, Bradley JA. Back to
the future: application of contemporary technology to long-
standing questions about the clinical relevance of human leuko-
cyte antigen-specific alloantibodies in renal transplantation. Hum
Immunol. 2009;70:563–8.
62. Mizutani K, Terasaki P, Hamdani E, Esquenazi V, Rosen A,
Miller J, et al. The importance of anti-HLA-specific antibody
strength in monitoring kidney transplant patients. Am J Transpl.
2007;7:1027–31.
63. Vaidya S, Partlow D, Susskind B, Noor M, Barnes T, Gugliuzza
K. Prediction of crossmatch outcome of highly sensitized patients
by single and/or multiple antigen bead luminex assay. Trans-
plantation. 2006;82:1524–8.
64. Wahrmann M, Exner M, Schillinger M, Haidbauer B, Regele H,
Ko
¨rmo
¨czi GF, et al. Pivotal role of complement-fixing HLA
alloantibodies in presensitized kidney allograft recipients. Am J
Transpl. 2006;6:1033–41.
65. Wahrmann M, Exner M, Haidbauer B, Schillinger M, Regele H,
Ko
¨rmo
¨czi G, et al. [C4d]FlowPRA screening–a specific assay for
selective detection of complement-activating anti-HLA alloanti-
bodies. Hum Immunol. 2005;66:526–34.
66. Smith JD, Hamour IM, Banner NR, Rose ML. C4d fixing, lum-
inex binding antibodies—a new tool for prediction of graft failure
after heart transplantation. Am J Transpl. 2007;7:2809–15.
67. Heinemann FM, Roth I, Rebmann V, Arnold ML, Witzke O,
Wilde B, et al. Immunoglobulin isotype-specific characterization
of anti-human leukocyte antigen antibodies eluted from explanted
renal allografts. Hum Immunol. 2007;68:500–6.
68. Arnold ML, Dechant M, Doxiadis II, Spriewald BM. Prevalence
and specificity of immunoglobulin G and immunoglobulin A non-
Clin Exp Nephrol
123
complement-binding anti-HLA alloantibodies in retransplant
candidates. Tissue Antigens. 2008;72:60–6.
69. Bartel G, Wahrmann M, Exner M, Regele H, Huttary N,
Schillinger M, et al. In vitro detection of C4d-fixing HLA
alloantibodies: associations with capillary C4d deposition in
kidney allografts. Am J Transpl. 2008;8:41–9.
70. Chin C, Chen G, Sequeria F, Berry G, Siehr S, Bernstein D, et al.
Clinical usefulness of a novel C1q assay to detect immunoglob-
ulin G antibodies capable of fixing complement in sensitized
pediatric heart transplant patients. J Heart Lung Transpl. 2011;30:
158–63.
71. Ho
¨nger G, Wahrmann M, Amico P, Hopfer H, Bo
¨hmig GA,
Schaub S. C4d-fixing capability of low-level donor-specific HLA
antibodies is not predictive for early antibody-mediated rejection.
Transplantation. 2010;89:1471–5.
72. Chen G, Sequeira F, Tyan DB. Novel C1q assay reveals a clin-
ically relevant subset of human leukocyte antigen antibodies
independent of immunoglobulin G strength on single antigen
beads. Hum Immunol. 2011;72:849–58.
Clin Exp Nephrol
123
