SPECIAL FEATURES
Consensus Guidelines on the Testing and Clinical
Management Issues Associated With HLA and
Non-HLA Antibodies in Transplantation
Brian D. Tait,1 Caner Süsal,2 Howard M. Gebel,3 Peter W. Nickerson,4 Andrea A. Zachary,5
Frans H.J. Claas,6 Elaine F. Reed,7 Robert A. Bray,3 Patricia Campbell,8 Jeremy R. Chapman,9
P. Toby Coates,10 Robert B. Colvin,11 Emanuele Cozzi,12 Ilias I.N. Doxiadis,6 Susan V. Fuggle,13
John Gill,14 Denis Glotz,15 Nils Lachmann,16 Thalachallour Mohanakumar,17 Nicole Suciu-Foca,18
Suchitra Sumitran-Holgersson,19 Kazunari Tanabe,20 Craig J. Taylor,21 Dolly B. Tyan,22
Angela Webster,9 Adriana Zeevi,23 and Gerhard Opelz 2,24
Background. The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of
human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by
complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter
referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the
clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led
to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require
resolution and careful consideration when interpreting antibody results.
The project was commenced in November 2011. A meeting of contributors
was held in Rome in May 2012 and the final manuscript was completed
in October 2012.
The members of the Antibody Consensus Group thank The Transplantation
Society for initiating and financially supporting this exercise. Additional
support was also provided by Deutsche Gesellschaft für Immungenetik
(G.O., C.S., and N.L.), The European Federation for Immunogenetics
(F.H.J.C. and S.V.F.), and the British Society for Histocompatibility and
Immunogenetics Association (C.J.T.).
The authors declare no conflicts of interest.
1
National Transplant Services, Australian Red Cross Blood Service,
Melbourne, Victoria, Australia.
2
Department of Transplantation Immunology, Institute of Immunology,
University of Heidelberg, Heidelberg, Germany.
3
Pathology and Laboratory Medicine, Emory University School of Medicine,
Atlanta, GA.
4
Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
5
Immunogenetics Laboratory, John Hopkins University School of Medicine,
Baltimore, MD.
6
Department of Immunohaematology and Blood Transfusion, Leiden
University Medical Center, Leiden, The Netherlands.
7
UCLA Immunogenetics Center, Department of Pathology, and Laboratory
Medicine, David Geffen School of Medicine, University of California at
Los Angeles, Los Angeles, CA.
8
Departments of Medicine and Laboratory Medicine, University of Alberta,
Edmonton, Alberta, Canada.
9
Centre for Transplant and Renal Research, University of Sydney, Westmead Hospital, Sydney, New South Wales, Australia.
10
Central and Northern Adelaide Renal and Transplantation Service,
Adelaide, South Australia, Australia.
11
Pathology Department, Massachusetts General Hospital, Boston, MA.
12
Unit of Clinical and Experimental Transplantation Immunology, University of Padua, Padua Medical Center, Padua, Italy.
Transplantation
& Volume 95, Number 1, January 15, 2013
13
Transplant Immunology and Immunogenetics, Oxford Transplant Centre,
Nuffield Department of Surgical Sciences, Oxford University Hospitals
NHS Trust, Oxford, UK.
14
Department of Medicine, University of British Columbia, Vancouver,
British Columbia, Canada.
15
Nephrology and Transplantation Department, Saint-Louis Hospital,
Paris, France.
16
HLA Laboratory, Charité Universitätsmedizin, Berlin, Germany.
17
Department of Surgery, Washington University in St. Louis, St. Louis, MO.
18
Department of Pathology and Cell Biology, Columbia University, New
York, NY.
19
Department of Surgery, Goteborg University, Goteborg, Sweden.
20
Department of Urology, Tokyo Women’s Medical University, Tokyo, Japan.
21
Tissue Typing Laboratory, Cambridge University Hospitals NHS Foundation
Trust, Addenbrooke’s Hospital, Cambridge, UK.
22
Histocompatibility Immunogenetics and Disease Profiling Laboratory,
Department of Pathology, Stanford University Medical Center, Palo
Alto, CA.
23
Department of Pathology, Pittsburgh Medical Center, Pittsburgh, PA.
24
Address correspondence to: Gerhard Opelz, M.D., Department of
Transplantation Immunology, Institute of Immunology, University of
Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg, Germany.
E-mail: Gerhard.Opelz@med.uni-heidelberg.de
G.O. initiated the project and contributed to the deliberations and writing of
the report. B.T.D. was responsible for coordination of the written material and writing of the report. All contributing authors were actively
engaged in discussions concerning the nature of the content and provided written sections for inclusion in the document. All authors have
indicated agreement with the final content of the paper.
Copyright * 2013 by Lippincott Williams & Wilkins
ISSN: 0041-1337/13/9501-19
DOI: 10.1097/TP.0b013e31827a19cc
www.transplantjournal.com
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
19
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www.transplantjournal.com
Transplantation
& Volume 95, Number 1, January 15, 2013
Methods. With this background, The Transplantation Society convened a group of laboratory and clinical experts
in the field of transplantation to prepare a consensus report and make recommendations on the use of this new
technology based on both published evidence and expert opinion. Three working groups were formed to address (a)
the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody
testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups
were established in November 2011 and convened for a ‘‘Consensus Conference on Antibodies in Transplantation’’ in
Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases
for this report.
Results. A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies
in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to
HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI
for antibody detection should be supplemented with cell-based assays to examine the correlations between the two
types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the
technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation
Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA
detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if singleantigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be
taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA
antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and
islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM
negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA
within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first
transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a
biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the
biopsy results.
Conclusions. A comprehensive list of recommendations is provided covering the technical and pretransplantation
and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are
intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for
HLA antibody detection when used in conjunction with traditional methods.
Keywords: Transplantation, HLA, Antibodies.
(Transplantation 2013;95: 19Y47)
uccessful transplantation in the mid-1960s was dependent on developing an understanding of humoral rejection that caused immediate loss of the kidney at the time
of transplantationVhyperacute rejection (HAR). The identification of antibodies to human leukocyte antigen (HLA)
antigens present on the graft and the subsequent development
of a simple and practical test for donor-specific antibodies
(DSA)Vthe complement-dependent lymphocytotoxicity
(CDC) cross-matching (XM) testVprovided the surgeon
and the patient with a reasonable basis for undertaking a
transplantation procedure (1).
Developments in technologies and our understanding
of antibody reactions since the 1970s have allowed refinement
of the methods laboratories can use to aid the prediction of
graft rejection. Recognition of autologous and non-HLA
antibodies, CDC XM techniques with increased sensitivity,
use of flow cytometry, and identification of IgM antibodies
with the aid of dithiothreitol (DTT) all provided a sophistication of assessment and development of more accurate prediction of which transplantations might and which might not
proceed safely.
Developments of immunosuppression medications in
the 1980s and 1990s centered on the control of T-cell
alloimmunity, and with increasingly successful and effective
protocols, the incidence of acute rejection fell considerably,
S
as did the rate of graft loss. This success has exposed our
relative lack of control over antibody-mediated or humoral
rejection processes and raised the relative importance of
both acute and chronic antibody-mediated rejection (AMR)
in graft loss.
Technical developments over the past 10 years have
also contributed to an increased understanding of alloimmune
biology. The first of these is the relatively reliable identification
of complement activation on the graft endothelial cell surfaces
using histologic localization of the complement component
C4d on transplant biopsies (2, 3). The second has been the use
of solid-phase immunoassays (SPI) to identify antibody specificity with precision and sensitivity (4, 5).
These new technologies challenge our established norms
but provide opportunities for further improvements through
application to clinical practice. The problem we now face
is that these technologies are highly sensitive, and we do
not fully understand the clinical impact of antibodies detected by these new methods. Current approaches aim at
risk stratification based on antibody identification and XM
results, taking into account the organ type and clinical considerations, such as urgency, available immunosuppression
strategies, and donor factors.
This consensus report is the product of the deliberations of three working groups addressing (a) the technical
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
+++
LUM SAB
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
21
The assays are scored from not valuable (j) to very valuable (+++). The comparative sensitivities are LUM9ELISA/FC9CDC. Note there are no convincing data that demonstrate ELISA is more
sensitive than FC.
AMR, antibody-mediated rejection; CDC, complement-dependent lymphocytotoxicity; ELISA, enzyme-linked immunosorbent assay; FC, flow cytometry; HAR, hyperacute rejection; HLA, human
leukocyte antigen; LUM, Luminex-based immunoassays (generic, phenotype, and single-antigen beads [SAB]); XM, crossmatch.
+++
+
+++
LUM phenotype
j
+++
LUM generic
j
+++
+++
ELISA generic
ELISA specific
j
j
+++
+++
Specification of HLA
antibodies
Comprehensive specification
HLA antibodies
+
++
Detection of antibody breadth
and level
Low level of sensitivity
j/+
Useful only if patient nonsensitized
Low level of sensitivity
j/+
++
Donor cells required
+
Donor cells required
j/+
Prevention of HAR or
+
early AMR
Prevention of HAR or
+
early AMR
Detection of HLA antibodies
+
Specification of HLA
(Only if patient sensitized)
antibodies
Detection of HLA antibodies
+
+++
+++
CDC/CDC
modified
FC/FC modified
Comment
Pretransplantation
XM
Pretransplantation
screening
Method
Solid-Phase Immunoassays
SPI obtained as commercially manufactured kits use
solubilized HLA molecules bound to a solid matrix that is
either a microtiter plate (enzyme-linked immunosorbent assay [ELISA]) or polystyrene beads (multiplexed multianalyte
bead arrays) performed on a conventional flow cytometer or a
small footprint fluoroanalyzer (Luminex) (11Y13). ELISA
results are expressed as optical density ratios compared with a
negative control, giving a semiquantitative assessment of antibody binding.
Bead-based array assays use polystyrene beads impregnated with different ratios of two fluorescent dyes
TABLE 1.
Comparison of Techniques
Cell-Based Assays
CDC and flow cytometry used for HLA-specific antibody screening and donor XM testing (6, 7) use cellular
targets. The CDC assay has lower sensitivity but identifies
antibodies that can mediate HAR (1). Technique modifications to increase sensitivity and specificity have been published but are not routinely used in all laboratories (8Y10).
The flow cytometry assay detects antibody binding to
target lymphocytes through a more sensitive method involving a fluorescent secondary antibody and quantification
via a flow cytometer. Flow cytometry XM (FCXM) represents
a risk but not necessarily a contraindication to transplantation. Modifications of the flow cytometry assay include the
detection of different immunoglobulin classes and subclasses,
differentiation of target cells, and Pronase treatment of
B-lymphocytes to reduce background nonspecific reactivity.
Methods for antibody screening and cross-matching in solid organ transplantation
TECHNICAL ASPECTS
The various assays for HLA antibody identification
differ greatly in the type of target, format, sensitivity, and
specificity. Accurate analysis and clinical interpretation of
the results requires both a high degree of expertise and experience and a knowledge of the patient’s immunologic
history, including sensitizing events and previous transplantation history. Assay targets may be either cells tested in
a cytotoxicity or flow cytometry assay or soluble antigens
tested in SPI. Because the details of these various assays are
widely available, this article will focus mainly on technical
highlights of the tests and factors that impact the results.
Basic information
pretransplantation
Posttransplantation
Comment
issues surrounding the use of SPI for antibody detection and
characterization, (b) the application of this as well as conventional technology in the pretransplantation setting, and
(c) the role of posttransplantation antibody monitoring.
The individuals who comprised each working group are
shown in the Appendix. Recommendations are made for the
application of current antibody technology in various clinical
settings, and suggested future directions in research are outlined. The recommendations are graded according to three
levels as follows: Level 1 indicates a procedure that ‘‘must’’ or
‘‘should’’ be performed based on published data and currently
proven practice; Level 2 suggests that a certain procedure is of
benefit, but when all the evidence is considered, the recommendation is not sufficient to assign Level 1; and Level 3 is a
consensus recommendation for which there may not be
published data but which the panel of experts deem to be
potentially of benefit.
Comprehensive locus/allele
specification
Tait et al.
* 2013 Lippincott Williams & Wilkins
22
www.transplantjournal.com
Transplantation
(classifier signals) to yield, theoretically, up to a 100 distinguishable bead populations. The antiglobulin reagent in the
bead assays is labeled with a third fluorescent dye (the reporter signal) so that, using a dual-laser instrument, the
fluorescence signature of each bead can be interrogated to
identify the bead population by one laser, whereas the reporter
fluorescence simultaneously assesses HLA-specific antibody
binding. The bead-based array assay is analyzed on the
Luminex platform and is semiquantitative. The level of HLAspecific antibody binding is expressed as the mean fluorescence intensity (MFI) of the reporter signal.
Three types of panels vary in the composition of their
target antigens: (a) pooled antigen panels have two or more
different bead populations coated with either affinitypurified HLA class I (HLA-A, HLA-B, and HLA-C) or HLA
class II (HLA-DR, HLA-DQ, and HLA-DP) protein molecules obtained from multiple individual cell lines and are
used as a screening test for the detection of HLA antibody;
(b) phenotype panels in which each bead population bears
either HLA class I or HLA class II proteins of a cell line derived from a single individual; and (c) single-antigen beads
(SAB) in which each bead population is coated with a molecule representing a single cloned allelic HLA class I or II antigen that enables precise antibody specificity analysis. Pooled
antigen panels are relatively inexpensive and indicate the presence or absence of antibody to a particular HLA class, but
they do not provide specificity nor do they represent all
possible antigens. However, these panels may be useful for
tracking changes in the level of HLA-specific antibody binding. Phenotype panels are similar to cell-based assays in
that more than one HLA specificity is present on each bead
population, which requires greater expertise in the interpretation of results than pooled or single-antigen panels. SAB
arrays are the most sensitive and specific, providing the
highest degree of HLA antibody resolution, and are particularly useful in the accurate identification of antibodies
in highly sensitized patients.
Numerous reports show varying degrees of correlation
between MFI, antibody level, XM results, and clinical outcomes (14Y16) but standardized cutoff values for positivity
have not been established. A comparison of the use of cellbased immunoassay versus SPI and their application in different types of organ transplants is found in Tables 1 and 2.
TABLE 2.
& Volume 95, Number 1, January 15, 2013
Advantages and Disadvantages of the Techniques
Complement-Dependent Lymphocytotoxicity
The indisputable advantage of the CDC assay for lymphocytotoxic panel reactive antibody (PRA) determination
and donor XM testing is the ability to predict (and therefore provide an opportunity to avert) HAR due to HLA DSA
(1, 17). Drawbacks are that the assay is not very sensitive,
requires a relatively large number of viable lymphocytes, and
can yield a positive result due to non-HLA antibodies. The
CDC method is difficult to standardize and assessment of
antibody breadth in waiting list patients may be confounded
by panel composition. Detailed specificity analysis using CDC
screening requires access to either large panels of local HLAtyped donors, as practiced in some laboratories (18Y20), or
the use of frozen commercial cell trays. Importantly, CDC
screening cannot distinguish all antibody specificities in highly
sensitized patients with complex antibody profiles.
Because the percent PRA is based on how many cells
give positive reactions (independent of specificity), the term
%PRA used as an indication of the level of a patient’s allosensitization can be misleading because centers with different cell panels are likely to achieve different PRA values
with the same serum (21). For these reasons and to more
accurately assess the probability of a positive XM due to
antibodies to either HLA class I or II, %PRA has been replaced
by ‘‘calculated reaction frequency’’, calculated PRA (cPRA),
or virtual PRA (22, 23).
Flow Cytometry
Although flow cytometry is also subject to reactions
caused by non-HLA antibodies, it is appreciably more sensitive than CDC and has been proven useful in identifying
patients with weak DSA who are at increased risk of AMR
and graft rejection (24). Flow cytometry assays are difficult
to standardize due to variability among cytometers, fluorochromes, antiglobulin reagents, and variations in cell-to-serum
ratios. The flow cytometry B-cell XM is associated with high
background antibody binding, which may be reduced by incubation of target lymphocytes with Pronase (25). However,
Pronase treatment may affect HLA expression and lead to
false-positive T-cell XM (26). Each center must therefore
validate FCXM result thresholds with respect to clinical risk.
Methods for antibody screening and cross-matching for each type of organ transplant
Organ/single or
combined
Kidney
Heart
Lung
Liver
Pancreasb/(islets)
Intestinal
Pretransplantation
screening
+++
+++
+++
+
+++
+++
Generic
methods
SPITCDC/FC
SPITCDC/FC
SPITCDC/FC
SPITCDC/FC
SPITCDC/FC
SPITCDC/FC
Extended methods
(SPI-SAB)
(All patients)
All patients
All patients
.
All patients
All patients
a
XM
Comment
a
CDC/FC/vXM
CDC/FC/vXMa
CDC/FC/vXMa
.
CDC/FC/vXMa
CDC/FC/vXMa
Prevention of HAR or
Prevention of HAR or
Prevention of HAR or
AMR
Prevention of HAR or
Prevention of HAR or
a
early AMR
early AMR
early AMR
early AMR
early AMR
Depending on the local/national/organ exchange organization policy.
Pancreatic islet cells can be transplanted with a positive XM.
Test methods to be employed according to the organ transplanted. In case of double transplants, the most stringent method should be used. The assays are
scored from not valuable (j) to very valuable (+++). The comparative sensitivities are LUM9ELISA/FC9CDC. Note there are no convincing data that
demonstrate ELISA is more sensitive than FC.
AMR, antibody-mediated rejection; CDC, complement-dependent lymphocytotoxicity; ELISA, enzyme-linked immunosorbent assay; FC, flow cytometry;
HAR, hyperacute rejection; SPI, solid-phase immunoassays; vXM, virtual crossmatch; XM, crossmatch.
b
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Tait et al.
* 2013 Lippincott Williams & Wilkins
TABLE 3.
23
Features of the Luminex SPI panels provided by the two vendors
Vendor A
Vendor B
Portfolio
Background assessment
Antigen sources
Antigen density report
Full automated solution
Special feature
Pooled, phenotype, and SAB. HLA class I and II,
MICA antigen beads. RUO and in part IVD
Three negative control beads. NC serum
Platelets, EBV-transformed lymphocytes. Blood donors,
recombinant cell lines
HLA class I and II and MICA
Available
DQ enriched beads in phenotype panels
Pooled, phenotype, and SAB. HLA class I and II,
MICA, and NA antigen beads. IVD
One negative control bead. NC serum
EBV-transformed lymphocytes, recombinant
cell lines
HLA class I
Available
EBV, Epstein-Barr virus; HLA, human leukocyte antigen; IVD, in vitro diagnostic; MICA, major histocompatibility complex class IYrelated chain A; NC,
negative control; RUO, research use only; SAB, single-antigen beads; SPI, solid-phase immunoassays.
Solid-Phase Immunoassays
ELISA technology is more sensitive than CDC (11),
whereas Luminex bead technologies are more sensitive than
both CDC and flow cytometry (12), enabling the detection
of low levels of HLA-specific antibody. The comprehensive
array of common and many rare HLA alleles for all 11 HLA
loci (A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1,
DPBA1, and DPB1) present in the Luminex SAB array
enables the precise definition of HLA antibodies contained
in complex sera (13, 27). The ability to identify epitope-specific
antibodies (28Y30) and antibodies to HLA-Cw, HLA-DQA,
HLA-DPA, and HLA-DPB was not previously possible in most
diagnostic routine laboratories and has led to a new realization
of the importance of such antibodies in kidney allograft rejection (31, 32).
SPI results are semiquantitative and enable broad categorization of DSA levels into low, intermediate, and high according to the optical density (ELISA), median channel of
fluorescence (flow cytometry), or MFI value (Luminex). Luminex phenotype and SAB panels provide large-scale batch testing results within 4 hr, making these tests valuable in supporting
TABLE 4.
a diagnosis of humoral rejection in routine pretransplantation
and posttransplantation monitoring and in assessing the efficacy of antibody reduction programs (33, 34). Table 3 displays
the features of the Luminex SPI panels provided by two vendors.
SPI, like CDC or flow cytometry, have technical aspects
requiring significant expertise in their use and interpretation
(Table 4). For SPI, it is relevant to capture both the HLA
antibody specificities identified and the level of antibody
(MFI). Immunologic risk assessment varies among centers,
ranging from listing SAB-defined specificities above a given
MFI threshold as unacceptable, listing antibody specificities
according to MFI range (low, intermediate, or high), or
providing MFI information for each defined antibody
specificity (27). It is important to note that although
Luminex-based assays can provide a semiquantitative numerical value, they were developed and licensed as qualitative assays.
Interface with Laboratory Databases
The complexity of the data obtained from SAB arrays,
particularly in highly sensitized patients, requires each
Technological advantages and limitations of Luminex HLA SAB
Technological advantages
Qualitative: enables precise identification of all antibody
specificities in complex sera (DSA)
Comprehensive: distinguishes antibodies to all common alleles
for HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3/4/5,
HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1
Semiquantitative: enables determination of antibody levels
(high, intermediate, and low)
Sensitive: enables detection of weak antibody levels
Rapid: enables real-time antibody monitoring for DSA. HLAi
transplantation. Pretransplantation and posttransplantation
antibody monitoring (assist diagnosis of AMR). Virtual XM
Enables detection of non-HLAYspecific antibodies (e.g., MICA)
Detection and differentiation between immunoglobulin class
and isotype (e.g., complement fixing and noncomplement
fixing C4d and C1q)
Technological limitations
Some positive results can be caused by antibodies to
denatured HLA.
Occasional high background binding requiring repeat testing
and absorption protocols.
Variable HLA protein density on beads. Blocking factors may
cause false-negative or misleading low assessment of
antibody levels (prozone?). IgM and C1 can block
IgG binding.
Lot-to-lot variation requiring validation. Vendor-specific
variation.
Reagents not standardized
AMR, antibody-mediated rejection; DSA, donor-specific HLA antibodies; HLAi, HLA incompatible; MICA, major histocompatibility complex class
IYrelated chain A; SAB, single-antigen beads; XM, crossmatch.
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
24
www.transplantjournal.com
laboratory to develop an interface between the Luminex
analysis software and the laboratory information system to
enable efficient and accurate analysis of antibody data.
Solid-Phase HLA Antibody Detection Assays:
Technical Challenges
Effect of Variability in Antigen Quantity and Quality
The relative quantity on beads of a particular antigen
differs substantially among pooled antigen, phenotype, and
SAB. HLA-Cw, HLA-DQ, and HLA-DP on SAB and DQ
on one manufacturer’s phenotype panel are characterized
by a higher relative antigen density. As a consequence, antibody levels to these antigens run the risk of being overestimated yet may represent only a low immunologic risk for
renal transplant rejection (27). Conversely, antibodies against
public epitopes such as Bw4 or Bw6 may appear underrepresented because a single antibody may be dispersed across
many beads underestimating its actual level. Disparities in
antigen quantity exist not only across the different bead formats but also among different HLA molecules on the SAB.
Recent improvements in the manufacturing process and
quality assurance measures have contributed to a more uniform antigen density across all beads from lot to lot, although
this problem has not been completely resolved. In addition,
the antibody analysis software available from the manufacturers includes features to normalize the data according to
an average antigen density. All these measures contribute to
more consistent results.
An issue inherent with the use of soluble HLA molecules is the fact that they are not in their native state and
environment. Proper conformation of HLA antigens depends
on the bound peptide and associated A2 m (class I) and is
affected by glycosylation pattern. Purification and coating to
the beads can lead to improper conformation of antigens that
gives rise to the detection of clinically irrelevant antibodies
(35, 36). Thus, deviations in the overall antigen condition
(quantity and conformation) due to different methods of preparation may lead to discrepant reactions between phenotype
and SAB.
Reports using SAB have suggested the existence of
naturally occurring HLA antibodies in males (37, 38), but
these antibodies appear to be specific for epitopes on denatured HLA molecules (38, 39). Tests of some of these
sera were shown to yield negative results in FCXMs, suggesting the absence of antibodies to HLA antigens in their
native conformation. Two recent reports have shown the
lack of clinical relevance of antibodies specific for epitopes
on denatured antigens (36, 40). Distortion of HLA molecules
that results from binding them to a solid matrix represents a
dual risk: interpreting positive reactions with cryptic epitopes
incorrectly as a contraindication to transplantation and failing
to recognize the presence of antidonor HLA antibodies because they do not bind to the distorted molecules. Zachary
et al. (41) demonstrated that the identity and level of DSA
using phenotype beads was more predictive for CDC and
FCXM results than were antibodies based on SAB testing.
Inherent Variability
As with any serologic assay, there is a certain degree of
inherent variability in SPI. This variability is seen among
Transplantation
& Volume 95, Number 1, January 15, 2013
different kits, different lots of the same kit, different runs,
and different operators. Manufacturers are making efforts to
reduce lot-to-lot variability and increase uniformity among
the beads within a kit. Users can reduce variability by strict
adherence to test conditions and procedures and by the use
of robotic liquid-handling instruments. The degree of variability is similar to that of cell-based assays. The identification
of strong antibodies is unequivocal and most discrepant
results observed with external proficiency schemes occur with
weak antibodies.
Interpretation
Reproducibility is a major prerequisite to facilitate the
proper interpretation of HLA antibody detection assays. In
assigning apparent specificity for DQ or DP, the possibility
of specificity for the > chain (DQA or DPA) and for epitopes specific to particular >-A chain combinations must be
taken into consideration (42). The interpretation of epitopespecific HLA antibody is very complex and requires personnel with appropriate experience and expertise. Most
importantly, the plausibility of antibody assignment must be
verified by considering the following: (a) consistency with
other antibody tests performed and with test results of other
specimens from the same patient, (b) serum donor phenotype
to ensure that a self-epitope is not included in the antibody
assignment, (c) alloimmunizing events (i.e., transfusion, pregnancies, or previous transplants), and (d) cross-reactivity. The
HLA phenotypes of the patient, previous organ donors, and,
in the case of known pregnancies, the paternal HLA antigens when obtainable should be considered to verify antibody
specificity. High-resolution HLA typing may be required because some patients can form antibodies to epitopes on
alleles included in their own antigen group. The HLA type
of the serum donor may also be used to define patient- and
serum-specific cutoff values. Reliable information on proinflammatory events of the patient is valuable in validating
sudden increases in antibody strength or breadth (43).
Assessment of Antibody Level
MFI levels on the beads represent the amount of antibody bound relative to the total antigen present on the beads
(degree of saturation), which varies by individual bead. MFI
does not represent titer despite the widespread misuse of the
term. The MFI has been used successfully in some centers as a
means of predicting flow cytometry or CDC XM results (44).
This approach is particularly useful in predicting negative XM
but may become less reliable with low levels of antibody. The
prediction of antibody level is particularly problematic with
strongly reacting antibodies. Some diluted sera give MFI
equivalent to that found in undiluted sera, indicating saturation of the specific antibody-binding epitope(s) on the HLA
molecule bound to the beads (45). In addition, some sera give
increased MFI on dilution due to interference by IgM or C1 in
undiluted sera (45Y47). The relationship between antigen
density on the beads and on cells is incompletely understood.
These factors compromise the use of MFI as a surrogate marker
of antibody level, the estimation of which requires serum dilution or other treatments to remove interfering substances.
Attempts have been undertaken to standardize MFI
by conversion to molecules of equivalent soluble fluorochrome
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
* 2013 Lippincott Williams & Wilkins
(MESF) using quantification beads known from flow cytometry (48). However, the inherent assay variability from day
to day, lot to lot, or among different laboratories could not be
eliminated by using MESF.
Accurate quantification of HLA antibody levels is required for therapeutic pretransplantation desensitization and
posttransplantation AMR rejection protocols. It has been
suggested that the relationship of any given bead to the positive
control bead may be useful in determining significant changes
or for normalizing MFI values (49, 50). It is suggested that
quantification of antibody level is best achieved by titration.
Interference in Solid-Phase Immunoassays
The reactivity due to substances other than the analyte
being tested and the reduction in the strength of reactivity with
the target analyte are problems inherent to serologic assays. The
development of SPI has increased exponentially the sensitivity
of tests for HLA antibodies; however, these tests remain susceptible to interference from a variety of substances (51, 52) that
may be categorized into two groups: substances present naturally
in serum and substances that are administered to patients.
Interference by Substances Inherent in the Serum
Removal or reduction of IgM from sera by hypotonic
dialysis increases the reaction strength of the positive control
bead, decreases reactivity with the negative control beads, and
affects both the strength and the specificity of antibodies
detected with the antigen-specific beads (53). Precipitation of
IgM in hypotonic dialysis might also trap immune complexes
that could bind nonspecifically to beads. Some of the interference is due to reactivity with the polystyrene beads as demonstrated by lack of inhibitory reactivity of some sera when
HLA antigens were bound to glass microchips (53). Dilution or
treatment of sera with DTT decreases the reactivity of some
antibodies and increases the reactivity of others (45); however,
it also increases the reactivity with negative control beads and
the strength of HLA antibodies from DTT-treated serum does
not appear to correlate well with XM results (53). Removal of
C1 via dilution, DTT, heat inactivation, or use of a C1 inhibitor
can restore masked HLA reactivity on SAB (46), suggesting that
the effect of DTT treatment is via cleavage of C1. It is
recommended that each laboratory decide under what conditions such treatments may be necessary. This may range from
pretreating all samples to having a specific indicator based on
testing parameters, such as a low positive control bead value.
Some reactivity with beads coated with HLA class I
molecules can be attributed to antibodies to HLA-E that
cross-react with the HLA classic class I molecules (54, 55).
The reactivity of the HLA-E antibodies is with HLA-B and
HLA-Cw, which does not explain the observed reactivity with
beads bound with denatured A-locus antigens (see previous
section on antigen quantity and quality).
The incidence of interference varies among different
groups of patients. For example, non-HLA antibodies that
are reactive in SPI assays appear after left ventricular assist
device implantation (56).
Interference by Exogenous Substances
Therapeutic reagents used to prevent or treat rejection
have been shown to cause interference in SPI for HLA
Tait et al.
25
antibodies. Among these agents are intravenous immunoglobulin (IVIg) given at high doses (2 g/kg body weight),
antithymocyte globulin, the proteasome inhibitor bortezomib,
and eculizumab, a complement C5 inhibitor. It is widely accepted that high-dose IVIg interferes in assays that use an antiglobulin reagent. Reactivity with the negative control beads in
SPI was reported to be increased more than fivefold after
treatment of patients with IVIg (57). Treatment with antithymocyte globulin results in apparent HLA-specific reactivity
(57Y59). Within days after treatment with either bortezomib or
eculizumab, sera from patients with antibodies yielding strong
reactions in SPI showed significant reductions in antibody
strength (57). However, after hypotonic dialysis of the sera,
there was either a very limited reduction or a slight increase in
antibody strength.
SPI are being used widely to guide treatment of transplant patients and to define unacceptable HLA antigens. Thus,
it is critical that interference in these assays be recognized and,
when possible, reduced or eliminated. Several patterns of reactivity that are indicators of interference are listed below.
Indicators, but not proof of interference in multiplexed microsphere assays include the following:
& High reactivity with the negative control bead,
& Low reactivity with the positive control bead,
& (Each laboratory has to establish what levels of positive
and negative controls indicate interference.)
& Sudden change in the pattern of reactivity in sequential
sera from a patient in the absence of any specific treatment
or event,
& Reactivity that does not reconcile with the results of CDC
or FCXM tests, and
& Reactivity with the patient’s own HLA antigens.
Modifications to Solid-Phase Immunoassays
for Detection and Assessment of Functionality
of HLA Antibodies
C4d Assay
The C4d and C1q assays are modifications to SPI designed to distinguish complement fixing from noncomplement
fixing antibody. The C4d assay (60Y63), which includes spiking
with normal human serum as the source of C4d, demonstrated
that, for class I and II HLA antibody detection, respectively, this
C4d assay has superior specificity compared with CDC (92/
100% vs. 79/86%) but inferior sensitivity (61/31% vs. 70/55%)
(61). The C4d assay requires complement activation to occur
and is influenced by complement regulatory factors. This
method has low sensitivity with maximal MFI values in the
3500 range. Clinical data obtained using various modifications
of the C4d assay have shown that the presence of C4d+ antibody correlates with graft survival in kidneys (62) and hearts
(63). C4d+ antibodies do not appear to be associated with AMR
in renal grafts (64), but an association with the presence of
C4d+ antibodies and C4d deposition in the peritubular
capillaries has been reported (62). More recent reports have
demonstrated that de novo DSA is associated with poor
patient survival but is independent of either IgG strength
(MFI) or the ability to fix C4d (65, 66).
C1q Assay
The C1q assay designed to distinguish complement
fixing from noncomplement fixing antibody does not
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26
www.transplantjournal.com
require complement activation other than the binding of
C1q to the antibody (67). It detects antibodies capable of
binding complement and initiating the classic pathway
irrespective of whether they do so or not. Thus, it is not
affected by complement regulatory factors other than, perhaps, C1INH (68). The method uses a standard amount of
exogenous purified hC1q added to the patient serum. The
C1q assay is highly sensitive, with maximum SAB MFI
values of more than 30,000. Although there is a trend for
higher MFI values detected by the IgG assay to fix complement, this is not uniformly true and prediction of the
complement fixing ability of a given antibody cannot be
made from the IgG results (67, 69). The C1q assay detects
more IgG antibodies than those detected by CDC but also
detects complement fixing IgM. In cardiac transplantations,
correlations have been demonstrated between antibodies
detected by the C1 q assay and AMR in the first month for
both preformed and de novo antibodies (70). Similar correlations with acute rejection and long-term graft outcome
have been observed in kidney transplant recipients (71, 72).
Analysis of epitopes by both IgG and C1q assays from pretransplantation through AMR resolution showed that C1q+
DSA correlated with the clinical course, whereas the IgG
reactivities did not (69). However, Otten et al. (73) found
no correlation with clinical course in kidney transplant
patients, but the frequency of C1q+ DSA was too low to
make meaningful conclusions. Although the C1q assay
shows promise, additional studies are required to establish
its clinical role as a routine test.
Detection of Antibodies to Non-HLA Antigens
Humoral responses to non-HLA antigens or tissuespecific autoantigens in the setting of solid organ transplantation are primarily to antigens expressed on endothelial cells
and epithelial cells. The incidence and clinical consequence of
immunization to non-HLA antigens is incompletely understood. A major limitation in our understanding of non-HLA
antibodies in transplantation is (a) the lack of knowledge of
the identity of the non-HLA targets and (b) a critical need for
the development of validated diagnostic screening assays for
direct detection of non-HLA antibodies to gain a better understanding of their clinical relevance.
AntiYendothelial cell antibodies (AECA) have been
reported to mediate endothelial cell activation, apoptosis,
and cell injury (74). AECA represent a heterogeneous group
of antibodies comprising both IgM and IgG isotypes and are
directed against a variety of antigenic determinants on endothelial cells (75). The endothelial cell is the principal
target for the detection of non-HLA antibodies involved
in AMR because it expresses antigens that are not present
on lymphocytes, which are typically used for the detection
of DSA. A major limitation in using endothelial cells as
a XM target is the lack of standardized protocols and
reagents, including positive and negative controls and
antiYendothelial cell reference sera. Historically, different
assay systems have been used to identify and characterize
AECA, including CDC (76, 77), flow cytometry (78Y81), and
immunofluoresence (82). These methods differ widely in their
sensitivity, specificity, and ability to detect distinct immunoglobulin isotypes. Another limitation is that the endothelial cells used for the detection and characterization of
Transplantation
& Volume 95, Number 1, January 15, 2013
AECA have been derived from third-party donors, not the
transplant donor. These laboratory-developed assays used
endothelial cells from different vascular beds, including microvascular (dermal), venous (umbilical cord), and large vessel
(aorta), which are known to have differences in protein expression resulting in distinct phenotypes (83Y85).
Surrogate endothelial cells can be useful for identifying antibodies to nonpolymorphic antigens or antigens with
limited polymorphisms. However, they have limited usefulness in detecting antibodies to highly polymorphic antigens, such as major histocompatibility complex class IYrelated
chain A (MICA), or to antigens that are uncommon or rarely
found in human populations. MICA is not constitutively
expressed on endothelium; rather, its expression is induced
under conditions of cellular stress. Given these caveats, SPI
using recombinant MICA protein targets represent a more
reliable detection system than primary endothelial cells. Ideally, once AECA are identified, the development of specific
SPI should facilitate their detection.
Two recent studies reported the study of AECA using
an indirect immunofluoresence method on commercially
available slides of human umbilical vein endothelial cells
(86, 87). A potential advantage of this method is that it is
commercially available and may permit standardization of
test results among laboratories using this as a screening test
to detect AECA.
A recent study (88) propagated endothelial cells
obtained directly from the transplant donor and used them
to study the development of posttransplantation endothelial
cell DSA after renal transplantation. Interestingly, these DSA
could be detected by cytotoxicity, suggesting an Ig isotype
that fixes complement (e.g., IgG1 and IgG3).
Antibodies can mediate graft injury via various mechanisms including complement-mediated injury, endothelial cell
activation, proliferation and apoptosis, and cellular recruitment (89, 90). Both complement activation and cellular
recruitment are regulated by the Fc portion of the immunoglobulin molecule, so it may be important to develop
assays that identify the isotype of AECA to understand the
pathogenesis of AMR. A recent study showed that donorreactive IgM AECA did not correlate with rejection, whereas
AECA of the IgG2 and IgG4 subclasses that do not activate
complement were enriched in recipients with rejection (91).
Lymphocyte XM tests fail to detect AECA. The XM-ONE
assay is an Food and Drug Administration (FDA)Yapproved
endothelial FCXM technique that uses endothelial cell precursor cells found in the peripheral blood at a frequency of 1%
to 2% (92). A benefit of this test is that it detects DSA and can
be used to test for antibodies to T lymphocytes, B lymphocytes,
and endothelial cells in the same assay (93). An important
question that needs to be addressed is whether AECA detected
using XM-ONE also bind to fully differentiated endothelial
cells that line the vessels of the allograft.
Discovery and Characterization of Antibodies
to Non-HLA Antigens
Proteomic approaches using protein extracts from
different sources, including cell lysates and protein microarrays, are being used for antibody screening and identification of specificities. There are several reports of protein
arrays for the discovery of non-HLA antigens that can
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
* 2013 Lippincott Williams & Wilkins
generate a humoral immune response after renal transplantation and for the discovery of alloantibody and autoantibody targets (94Y97). Two-dimensional immunoblotting
of IgG from patient sera has also been used for the identification of AECA (98). Antibodies to non-HLA targets detected
after lung transplantation have been described using a novel
technique called SEREX (99).
There have been a variety of non-HLA targets identified including MICA (81, 100, 101), vimentin (102Y106),
angiotensin II type 1 receptor (107, 108), tubulin (109, 110),
myosin (111, 112), and collagen (113, 114).
Toward Standardization of Methods
Solid-phase HLA antibody assays are complex and
variability in manufacturer reagents, assay performance, and
data analysis are barriers to harmonization of this assay
across laboratories worldwide. It is recommended that laboratories and kit manufacturers standardize critical components that have the potential to influence the results and
interpretation of SPI, including the following: HLA source
and preparation method, panel composition, and appropriate allele coverage, including DQA1, DPBA, and DPB1.
Other considerations for standardization are the antigen
density on the bead, antigen integrity (e.g., denatured vs.
native antigen), and the anti-human immunoglobulin detection reagentsVall critical factors that impact assay performance and interpretation. Technical variance can be
reduced by using the same standard operating procedure
including the type of plastic trays used (V-bottomed vs. Ubottomed), the serum volume to bead ratio, washing methods
(e.g., spin/flick vs. filter tray), vortexing methods, and employing a unified approach to sample preparation to minimize
interference in the assay (53, 115). If affordable, laboratories
should be encouraged to use automated laboratory equipment to achieve uniformity in dispensing reagents and assay
washing steps. In addition to optimizing protocols and reagents, fluoroanalyzers and flow cytometers should be calibrated using control particles to achieve similar target values
across instruments.
Cell-based antibody detection assays including CDC
and flow cytometry also differ widely among laboratories
worldwide (21). For cooperative studies, or studies attempting to produce comparable data, standardization is necessary.
The major areas of standardization include harmonization of
standard operating procedures including cell isolation, cellto-serum ratio, incubation, and wash steps. It is important to
define the monoclonal antibodies used for defining T and B
lymphocyte populations, secondary antibodies used for the
detection of human IgG and negative/positive controls. In
addition, instrument setup and data analysis are crucial variables as well. Periodic assessment of the degree to which
results are reproducible within a laboratory should be part of
the ongoing quality control of every laboratory.
Efforts should be made to standardize the interpretation and reporting of test results using defined reporting
algorithms, background normalization, and the potential
use of standard fluorescent intensity and MESF to compensate for Luminex and flow cytometer machine differences, respectively. For SPI, it is recommended that test
reports include the following critical values: assay type,
cPRA, antibody specificity, interpretation of antibody level,
Tait et al.
27
comments on presence or absence of DSA, criteria for positive/negative results, immunoglobulin isotype (e.g., IgG vs.
IgM), and any factors that are considered to affect test
values and interpretation of results.
A repository of well-characterized HLA polyclonal and
monoclonal reference reagents for ongoing technique and
reagent validation, monitoring interlaboratory variability,
and reproducible quantification of fluorescence values should
be developed. This should encompass a diverse set of reference sera to all HLA class I and II antigens representing different titers and isotypes. The reference reagents must be
validated in national and international exchange and on cell
panels and SPI for all available techniques. Each laboratory
must participate in relevant external proficiency testing programs as required by local, regional, and national regulations.
ANTIBODY TESTING
PRETRANSPLANTATION
In this section, the pretransplantation challenges encountered in the determination of clinically relevant alloantibodies in sensitized patients, the available options for the
timely and successful transplantation of patients with high
levels of alloantibodies, and the impact of sensitization against
HLA as well as non-HLA antigens on the outcome of kidney
and other organ transplants are discussed.
Determination of Unacceptable HLA Antigen
Mismatches and Risk Assessment in
Kidney Transplantation
A major task of HLA laboratories is the determination
of the so-called unacceptable HLA antigen mismatches
(UA). Using this information, negative XM prediction or
‘‘virtual XM’’ is possible when a potential donor’s complete
HLA typing is available. The determination of UA is a critical decision step because the likelihood of an organ offer
diminishes with increasing number of UA and all too frequently patients die on the waiting list before they can be
transplanted. Conversely, unrecognized UA, due to insensitive or incorrect testing, result in inferior graft survival and
futile organ shipments because the XM test in the recipient
center is positive.
The introduction of SAB assays has allowed for the
precise determination of UA not previously possible, especially in highly sensitized patients (13). Assignment of UA
should not generally be based on the SAB reactivity alone
but whether the antibody reactivity pattern is consistent
with a recognized epitope and the patient’s history of sensitizing events. Many centers still consider repeat HLA
mismatches from previous transplantations as UA even if
they give negative results in antibody screenings.
With the advent of more sensitive antibody assays, it is
presently unclear which antibody test at what sensitivity level
is most appropriate for the determination of UA. Donorspecific IgG HLA antibodies detected by CDC are considered
a contraindication for transplantation, whereas DSA detected
by other assays represent varying degrees of risk (116). Many
laboratories no longer use CDC assays to screen for HLA
antibodies and therefore depend on SPI alone to determine
whether an antigen is unacceptable. Although there are good
data for kidney transplants and other organs that preexisting
DSA by SPI is associated with an increased risk of rejection,
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
28
www.transplantjournal.com
usually AMR, and inferior outcomes (Tables 5 and 6), it is
debatable whether the antibodies that go undetected in CDC
and ELISA and are detectable exclusively in SPI bead assays
influence outcome (24, 31, 34, 73, 117Y134) (Table 5). Currently, individuals with multiple antibody specificities on SAB
testing but negative in CDC testing make up a large part of
the transplant waiting list. Many recipients with DSA positive
only by flow-based or Luminex technology do well posttransplantation and have good long-term graft function
(Table 5). Therefore, it is difficult to set a cutoff for acceptable risk. One should also consider that a low MFI might be
due to sera being screened at a time remote from the original
immunizing event, and in the absence of historical sera, the
characteristics of previous more highly reactive sera are unknown. A prospective XM with donor cells and historical serum is frequently not available at the time of organ offer and
there is no good test to determine which antigens are likely
to trigger an amnestic response. Furthermore, the MFI often does not correlate with strength of XM, indicating that
immunologic risk cannot be determined based on this parameter alone.
DSA that persist posttransplantation after desensitization therapy are considered a risk factor for developing
transplant glomerulopathy (TG) and subsequent graft loss
(119, 135). To truly understand the impact of bead assaydetected pretransplantation DSA, both short-term and longerterm outcomes need to be documented. In addition, these
outcomes may be impacted by desensitizing protocols and
induction with depleting agents that were not always given
to all recipients, so using retrospective data to answer all
these questions is often fraught with difficulty.
There are now data that indicate an increased risk of
AMR when the transplantation is performed in the presence
of DSA (Table 5). Many transplantation programs do not
want to transplant across a bead-positive DSA, and as many
programs allocate based on a virtual XM, the recipient may
be excluded based on the bead result without the opportunity to be cross-matched with donor cells. Most data are
from retrospective studies with variable immunosuppressive
regimens, so it is difficult to get a true estimate of risk because no control groups exist.
Transplantation of Highly Sensitized Patients
The old dogma that the presence of DSA pretransplantation is a contraindication for transplantation was the
reason that highly sensitized patients accumulated on the
waiting list because the serologic XM with almost all donors
was positive. The introduction of the more sensitive SPI has
led to an increase in the number of highly sensitized patients
but also to the knowledge that the presence of DSA is not always a contraindication but rather a risk factor. The risk for
rejection and graft loss can be decreased in two ways: (a) selection of a donor toward whom the patient has no DSA or
(b) removal of the DSA via desensitization protocols.
Strategies to Transplant the Highly Sensitized Patients
Special programs are necessary to increase the chance
that a highly sensitized patient can be transplanted with
a XM-negative donor without desensitization. For patients
waiting for a deceased donor organ, the acceptable mismatch
Transplantation
& Volume 95, Number 1, January 15, 2013
program of Eurotransplant has been shown to be very efficient (18). The basis of the acceptable mismatch program is
the precise identification of those HLA antigens toward
which the highly sensitized patient did not form antibodies.
Similar programs exist internationally where highly sensitized patients are prioritized to receive virtual XM-negative
organs. Patients with an incompatible living donor can participate in a paired donor exchange program. In cases where a
sensitized patient has a living donor available toward whom
the patient has formed DSA, paired donor exchange programs can facilitate transplantation with an alternative XMnegative donor.
If a compatible donor is not identified, desensitization
can be performed in combination with other measures.
Desensitization protocols aim to lower DSA at the time of
transplantation to a threshold considered safe and to maintain
the DSA at this level, also with the help of immunosuppression, at least for the first days to weeks after transplantation.
Hereby, alloantibodies are removed from the patient’s circulation by plasmapheresis or immunoadsorption and their
production is suppressed by T-cell immunosuppression, IVIg,
rituximab, or the proteasome inhibitor bortezomib based on
the rationale that depletion of B lymphocytes or plasma cells
may reduce DSA production. An additional approach is the
blockage of complement activation by administration of the
complement C5 inhibitor eculizumab.
Unfortunately, randomized controlled trials that compare the clinical efficacy of different desensitization strategies
are missing. Desensitized patients generally receive more
potent immunosuppression than DSA- or XM-negative sensitized patients. It is a matter of debate whether desensitization prevents de novo HLA alloantibody production and
results in good long-term outcome. Montgomery et al. (136)
reported recently that, compared with waiting for a compatible deceased donor, desensitization is an option for the
timely transplantation of sensitized patients with DSA. In this
study, living-donor organ transplantation after desensitization provided a significant survival benefit for patients with
HLA sensitization (136). Increasing evidence suggests that, in
patients carefully selected based on antibody titers, desensitization can be performed safely with good graft and patient
survival in living-donor as well as deceased-donor transplantation (137, 138), and a combination of desensitization, acceptable mismatch program, good HLA matching, and other
measures in an integrative manner is reported to result in a
significant reduction in waiting time and good allograft and
patient survival (139).
Impact of Sensitization Against HLA on Outcome
of Transplants Other Than the Kidney
As in kidney transplantation, cross-matching is a routine procedure also in pancreas transplantation. Although the
determination of UA and virtual cross-matching have become routine procedures at an increasing number of heart
and lung transplant centers in recent years, in other organ
and islet cell transplantation, more data are required to establish the precise role of pretransplantation DSA on graft
outcomes. A selection of published literature on the impact
of preexisting HLA antibodies in heart, lung, liver, pancreas,
intestine, multivisceral organ, and islet cell transplantation is
shown in Table 6 (70, 117, 140Y183).
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Literature on the impact of SAB-detected preexisting DSA on kidney transplantation outcome
First author
(reference)
Year n
Bryan (117)
Gibney (118)
Patel (119)
Burns (120)
Eng (121)
Van Den Berg-Loonen (122)
Aubert (123)
Vlad (124)
Phelan (125)
2006
2006
2007
2008
2008
2008
2009
2009
2009
DSA+ (n)
DSA loci
Method
XM
Donor
Preselection (No.)
AMR
CR
GS
10
136
330
70
83
37
113
355
64
10
20
21
70
27
13
11
27
12
AB
ND
AB DRDQ
Class I and II, no details
AB DRDQB
AB DRDQ
AB DRDQDP
ABC DRDQ
ABC DRDQ
F-PRA/SAB
LSC
LSC/F-PRA
LSC/SAB
LSC/SAB
SAB
LSC/SAB
LSC/SAB
LSC/SAB
AHG-CDC, FCT
AHG-CDC
CDC and FC T&B HI
FC T&B
CDC
CDC
CDC
CDC
CDC
DD
LD/DD
LD
LD (2 DD)
DD
DD
LD/DD
DD
LD
30% but no control
j
j
j
j
ND
6
j
6
ND
ND
6
ND
6
ND
ND
ND
6
6
,
ND
ND
,
6
6
6
6
Gupta (126)
Amico (127)
Riethmuller (128)
2009 121
2009 334
2010 155
16
67
20
ND
AB DRDQDP
AB DRDQ
LSC/SAB
SAB
LSC/ELISA/SAB
CDC
CDC T&B
Luminex
ND
LD/DD
LD
6
j
j If class I DSA
6
ND
ND
6
, If AMR
ND
Singh (129)
Gloor (130)
Lefaucheur (131)
SAB
LSC/SAB
ELISA/SAB
CDC
FC T&B
CDC
DD
LD/DD
DD
j If class II DSA
j
j
ND
j If AMR
ND
, If DR DSA
, In XM+
,
F-PRA/SAB
LSC/SAB (most sera)
CDC
CDC/FC
LD
LD/DD
F-PRA+
Desensitized
j
j
j
NS
NA
,
Susal (133)
2010 237
159
AB DRDQ
2010 189
117 (12 XMY)
AB DRDQ
2010 402 83/76 at time of
Class I and II no details
transplantation
2011 34
22
AB DR
2011 112 84 (17 CDCXM+, 44 ABC DRDQDP
FCXM+, 23
only SAB)
2011 236
53
ABC DRDQAB DPAB
CDC XM+/AHG XMj
No
FCj
Desensitized
B-cell CDC+
AM program
Thymo for high risk
No
Some had prospective
FCXM; ELISAj
No
No
Some had rituximab or
ATG for high risk
No
XM+ vs. XMNo
SAB
CDC
DD
ND
ND
NS
Dunn (31)
Couzi (24)
Couzi (24)
Caro-Oleas (134)
Otten (73)
2011 587
2011 45
2011 45
2012 892
2012 837
AHG-CDC T&B
FC T&B
FC T&B
CDC
AHG-CDC, FCB
LD/DD
DD
DD
DD
LD/DD
j
j
6
ND
ND
j
,
ND
6
ND
6
ND
,
ND , If DSA I+ and II+
Ishida (132)
Higgins (34)
46
ABC DRDQAB DPAB
LSC/SAB
30 historic (28 D0) ABC DRDQDP
LSC/ELISA/SAB after 2005
11
ABC DRDQDP
LSC/ELISA/SAB after 2005
103
ABC DRDQDP
LSC/SAB
290 (30 C1q+)
ABC DRDQ
SAB/C1qSAB
CDCj and ELISAj;
3-yr GS
Thymo
FCXM+
FCXMThymo for high risk
No
Tait et al.
AHG, anti-human immunoglobulin; AM, acceptable mismatch program; AMR, antibody-mediated rejection; CDC, complement-dependent lymphocytotoxicity; CR, cellular rejection; DD, deceased donor;
DSA, donor-specific HLA antibodies; F, flow; FC, flow cytometry; GS, graft survival; HI, highly immunized; LD, living donor; LSC, Luminex screen; NA, not applicable; ND, not determined; PRA, panel reactive
antibody; SAB, Luminex single-antigen beads; XM, crossmatch; j, increased; ,, decreased; 6, no difference.
* 2013 Lippincott Williams & Wilkins
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
TABLE 5.
29
30
Literature on the impact of preexisting HLA antibodies on nonkidney transplants
First author
(reference)
Year
n
DSA+ (n)
XM
Preselection
AMR
CR
GS
AB DR
ND
ND
ND
ND
ND
ND
ND
ND
ND
AB DRDQ
ND
ND
FCXM
FCXM,
FCXM
F-PRA
ELISA
CDC, F-PRA
CDC, ELISA
NA
NA
LMix, SAB, C4d
CDC
F-PRA
F-PRA, CDC, Luminex
FC
CDC
CDC
No
No
Rejection
Ped
Ped
Presensitized
No
No (1990-99)
No (2000-07)
No
No
Ped
Ped
j In class I
ND
j
6
j
ND
ND
ND
ND
ND
j
6
6
j In class II
ND
j
ND
6
ND
j
ND
ND
ND
ND
ND
6
,
,
ND
,
ND
,
6
6
,
,
6
,
,
ND
AB DRDQ
CDC, F-PRA
CDC, SAB
ABC DRDQ
Preexisting HLA antibodies in lung transplantation
Lau (154)
2000
200
18
Appel (155)
2005
380
Class I=9, class II=4
Hadjiliadis (156)
2005
656
Appel (157)
2006
341
Shah (158)
Stuckey (159)
2008
2011
10,237
1
Mangi (160)
2011
481
Preexisting HLA antibodies in heart transplantation
Bishay (140)
2000
338
Class I=50, class II=144
Bishay (141)
2000
500
PRA 910% 53
Michaels (142)
2003
44
12
Jacobs (143)
2004
60
8
Di Filippo (144)
2005
45
12
Leech (145)
2006
262
35
Feingold (146)
2007
168
23
Opelz (147)
2009
7797
347
Opelz (147)
2009
2326
98
Rose (148)
2009
565
19 (11 were C4d+)
Ho (149)
2009
774
Class I=71, class II=104
Scott (150)
2011
83
12 (PRA 925%)
Mahle (151)
2011
1904
397 (PRA 910%), 189
(PRA 950%)
Kobashigawa (152)
2011
523
95
Gandhi (153)
2011
85
34
Chin (70)
2011
18
Preexisting HLA antibodies in liver transplantation
Fung (161)
1988
12 KL
Muro (162)
2005
254
Mosconi (163)
2006
1 KL
Castillo-Roma (164) 2008
896
Reichman (165)
2009
1 KL
CDC, FC
CDC
CDC
CDC
CDC, Luminex
CDC
CDC
NA
No
No
6
j
ND
j
6
ND
SAB
NA
CDC-AHG, FC,
Virtual
Virtual
Ped
In C1q+
ND
ND
ND
ND
PRA
PRA
NA
NA
No
Desensitized
ND
ND
j
,
ND
37 (910%), 20
(925%)
19
ND
CDC
NA
No
ND
ND
CDC, PRA
NA
ND
ND
ND
240 (925% PRA)
1
ND
ND
CDC, PRA
PRA
NA
NA
ND
ND
ND
ND
,
ND
NA
Mainly for comparing
CDC and PRA
No
Case report, successful
depletion of antibodies
with good outcome
No
j
ND
ND
CDC
CDC
CDC
CDC
CDC
No
No
No
No
No
6
6
6
ND
j
ND
6
6
j
ND
6
,
ND
,
,
13 (PRA 920%),
3 C1q+
136 (class I PRA
910%), 64
(class II
PRA 910%)
4
14
1
89
1
ABC DRDQ
ND
AB DR
AB DR
AB DR
AB DRDQ
CDC, ELISA, PRA
NA
F-PRA
CDC
LMix
SAB
After
desensitization
ND
,
& Volume 95, Number 1, January 15, 2013
Method
Transplantation
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
DSA loci
www.transplantjournal.com
TABLE 6.
Cardani (182)
2007
Naziruddin (183)
2011
66 (40 I, 17
IAK, 9 SIK)
303
7/66 (6 I-BMT),
1-ITA
NA
AB DR
ND
NA
NA
SAB
ELISA
SAB
SAB
SAB
PRA
SAB
CDC T
CDC T
CDC
CDC
NA
CDC
NA
CDC FC
CDC
No (1990-99)
No (2000-07)
No
No
No
No
No
No
No
ND
ND
6
ND
j
j
ND
ND
ND
ND
ND
6
j
j
ND
j
ND
j
,
,
,
ND
ND
ND
ND
,
ND
F-PRA
SAB and dilutions
FC-T, AHG-B
AHG-T FC-T
4/10 FC-T positive XM
Case report
3/10 (1 SPK)
Yes
ND
ND
6
ND
F-PRA
CDC-PRA
Flow
ELISA, SAB
ELISA, SAB
SAB
CDC
CDC
CDC FC
CDC
CDC
NA
No
No
No
No
No
No
ND
ND
j
j
ND
ND
j
ND
ND
j
j
j
,
,
ND
,
ND
ND
ELISA, F-PRA
CDC, FC T&B
No
ND
ND
,
F-PRA, FSA
AHG-CDC,
FC T&B
CDC
No
ND
ND
,
No
ND
ND
NS
ND
ND
6
ELISA, LSC
27 centers CDC,
ELISA, F-PRA
and Luminex
NA
PRA used as a surrogate
for sensitization
Tait et al.
All studies were retrospective; data on survival and rejection not available.
AHG, anti-human immunoglobulin; AMR, antibody-mediated rejection; BMT, bone marrow transplantation; CDC, complement-dependent lymphocytotoxicity; CR, cellular rejection; DD, deceased
donor; DSA, donor-specific HLA antibodies; F, flow; FC, flow cytometry; GS, graft survival; HI, highly immunized; I, intestine alone; IAK, islet and kidney; ITA, islet transplantation alone; KL, combined
kidney-liver transplantation; KP, combined kidney-pancreas; L, liver; LD, living donor; LMix, Luminex mix; LSC, Luminex screen; M, multivisceral; NA, not applicable; ND, not determined; Ped, pediatric;
PRA, panel reactive antibody; SAB, single-antigen beads (Luminex or flow); SIK, sequential islet and kidney; XM, crossmatch; j, increased; ,, decreased; 6, no difference.
* 2013 Lippincott Williams & Wilkins
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Opelz (147)
2009
4518
443
ND
Opelz (147)
2009
2645
153
ND
Goh (166)
2010
139
33
AB DR
Girnita (167)
2010
73
21
AB DR
Musat (168)
2010
43
17
AB DR
Dar (169)
2011
6 KL
6
AB DRDQ
O’Leary (170)
2011
39
39
AB DR
Askar (171)
2011
2484
PRA 30%
ND
Lunz (172)
2012
809
100
AB DRDQ
Preexisting HLA antibodies in pancreas transplantation
Bryan (117)
2006
10 (1 KP)
7
AB
Melcher (173)
2006
1
1
ABC DRDQ
Preexisting HLA antibodies in intestine and multivisceral transplantation
Kato (174)
2006
I 6, M 21
ABDR
Sindhi (175)
2010
I 103
ABDR
Ruiz (176)
2010
I1
1
AB DR
Wu (177)
2010
I 134, L+I 76
53
AB DRDQ
Ashokkumar (178)
2010
I 70
ND
ABC DRDQ
Tsai (179)
2011
I 4, M 11
9
AB DR
Preexisting HLA antibodies in islet cell transplantation
Mohanakumar
2006
7/12
3/7
AB DR
(180)
Campbell (181)
2007
81/151
11
ABC DRDQ
31
32
www.transplantjournal.com
Although there are no randomized clinical trials giving
clear evidence for the impact of preexisting antibodies on heart
transplantation outcome, several reports strongly suggest that
allosensitization in patients awaiting heart transplantation remains a significant problem largely due to increasing rates of
sensitization that result from the administration of blood
products after the insertion of assist devices and, in pediatric
patients, after the use of allograft tissue in congenital heart
surgery (150, 151). Sensitization, in particular, the presence
of DSA, has been shown to be associated with a higher incidence of rejection and inferior graft outcome (70, 140Y153)
and there is evidence that complement fixing DSA are detrimental for graft outcome (70, 148). Recent reports indicate
that the successful depletion of alloantibodies can lead to
transplantation of highly sensitized heart recipients without
HAR (145, 152).
There are reports of HAR after lung transplantation
across positive XM. Therefore, preexisting antibodies to
HLA are generally viewed as detrimental in lung transplantation (154Y157, 160). Although no evidence is available on
the impact of PRA on clinical outcome, a large study reported
that PRA of more than 25% has significant negative prediction toward successful functioning of lung allografts (158),
and in two other studies, pretransplantation desensitization
therapy has been associated with improvement in clinical
parameters, including acute rejection and bronchiolitis obliterans (155, 159).
In liver transplantation, the impact of allosensitization
on outcome is still controversial, and pretransplantation
HLA testing and cross-matching are currently not routine
procedures. More recent studies, however, indicate that pretransplantation cross-matching may be relevant in the setting
of liver transplantation and that especially a positive T-cell
XM and the presence of pretransplantation DSA are associated with poor graft survival (147, 162Y170, 172). Preformed
HLA antibodies were also associated with poorer survival of
retransplants (166). Lunz et al. (172) found in a recent study
that acute cellular rejection was more common in patients
who were DSA and XM positive and demonstrated C4d
positivity in the biopsies of these patients as early as 3 weeks
after transplantation.
In combined liver-kidney transplantation, it has been
proposed that the liver will offer immunologic protection
against rejection of the kidney allograft (161). However,
recent studies including a large analysis of over 2484 combined liver-kidney transplant recipients indicate that presensitization has a negative impact on both overall patient
survival and kidney graft survival (163, 165, 169, 171). Although in sensitized recipients of combined liver kidney
transplants the liver was thought to be protective and prevent AMR in the presence of preformed HLA antibodies
(161), in more recent studies, preformed DSA were reported
to promote AMR in the kidney (165) and it was shown that,
in sensitized patients with preformed class II DSA, the liver
may not be fully protective (169).
There are limited data on the impact of pretransplantation
antibodies on pancreas graft function. Most pancreas transplants
are performed as simultaneous pancreas-kidney transplants
and the data in this group have mostly focused on the kidney
because the pathologic criteria for AMR in the kidney were
more clearly defined (117). Early graft loss from thrombosis
Transplantation
& Volume 95, Number 1, January 15, 2013
is not uncommon in pancreas transplantation, but hyperacute or accelerated acute rejection may not be considered a
factor in early graft loss, and as a result, the impact of preexisting antibody against donor HLA may have been underestimated (173). The criteria for diagnosis of AMR in the
pancreas have only recently been established. This will allow
for better diagnosis of AMR and will lead to clarification of
the role of antibody in the risk for AMR and impact on graft
outcomes (184).
Data on intestinal transplantation are limited. In isolated intestinal allografts, preformed HLA antibodies have
been shown to have significant adverse effects on allograft
survival (174, 175). Furthermore, a case of immediate AMR
was also documented in an intestinal transplant with DSA
and positive XM (176), and pretransplantation and posttransplantation de novo DSA were significantly associated
with the frequency and severity of acute cellular rejection
(177Y179).
Whereas two studies reported that pretransplantation
DSA detected by flow beads and or CDC were associated
with inferior transplant survival of islet cell transplants (180,
181), in another study, the same association could not be
found conceivably because the patients were often transplanted in conjunction with a kidney or received donor
bone marrow with the islet infusion (182). In a multicenter
registry report, pretransplantation PRA (DSA was not determined) was shown not to be predictive of islet cell
transplant failure, whereas posttransplantation PRA was
associated with poor outcome (183). Although there are few
data available, the studies that measured DSA in islet cells
alone recipients showed a negative impact of preexisting
HLA antibodies.
Impact of Pretransplant Non-HLA Antibodies on
Organ Transplantation Outcomes
Although, as shown in Table 7, several groups reported
on a possible impact on transplantation outcomes of pretransplantation antibodies against non-HLA targets, such as
MICA, endothelial cell antigens, islet cells antigens, collagen,
K->1 tubulin, cardiac myosin, and vimentin, at present, there
are no studies giving clear evidence for a strong role of such
non-HLA antibodies in solid organ or islet cell transplantation (91, 92, 108, 111, 112, 185Y212). An important group
of non-HLA antibodies are the isoagglutinins to ABO blood
group antigens. Traditionally, ABO-incompatible transplants
have been avoided but currently numerous living-donor transplants are performed across blood group mismatches, and
many studies indicate that conventional immunosuppression
can achieve comparable outcomes to ABO-compatible transplants provided that isoagglutinin titers are reduced pretransplantation (192, 193, 196, 197, 206).
In kidney transplantation, results obtained in a large
cohort of patients from the Collaborative Transplant Study
Group (187) indicated that, even if the donor was an HLAidentical sibling, a significant number of patients with high
panel reactivity pretransplantation had lower graft survival,
suggesting a role for immune responses to non-HLA antigens
in allograft rejection. The presence of preexisting antibodies to
MICA has been shown to correlate with kidney graft outcome
in some reports (186, 188, 194, 198, 199) but not in other
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
First author
(reference)
Impact of preexisting non-HLA antibodies on organ transplantation outcomes
Year
n
Antibody+ (n)
Method
Preselection
AMR
CR
GS
EL
AECA
MICA
Non-HLA
MICA
AECA
AECA
AECA
AECA
ABO
ABO
AT1R
MICA
AT1R
AECA
ABO
ABO
MICA
MICA
ELISA
FC
PRA
Luminex
FC
Cell-based ELISA
FCXM-ONE
Cell-based ELISA
Tube
Latex Agglutination
ELISA
Luminex
ELISA
FC
Tube
Tube
Luminex
FC
No
No
HLA-id
No
Case Report
No
No
No
ABO-i
ABO-i
No
Case report
No
No
ABO-i
ABO-i
No
No
j
j
ND
j
j
j
j
j
6
j Only if titer 91:32
j
j
j
j
6
6
ND
6
ND
ND
ND
ND
ND
ND
ND
j
ND
ND
ND
ND
ND
ND
ND
ND
j
ND
ND
,
, At 10 yr
,
ND
ND
ND
,
6
6
ND
ND
,
ND
6
ND
ND
6
II-2
II-2
II-2
II-2
II-2
II-2
II-2
II-2
II-2
II-2
II-2
II-3
II-3
II-2
II-2
II-2
II-2
II-2
Myosin
Myosin
Myosin
MICA
MICA
ABO
Non-HLA
MICA
ABO
SDS, Western blot
ELISA
ELISA
CDC, Luminex
CDC, Luminex
NA
CDC, Luminex
Luminex
Agglutination
No
Yes, patients with DCM
No
No
No
ABO-I Ped
No
No
Ped
j
j
ND
6
6
6
ND
6
6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
,
ND
ND
6
,
j
6
II-2
II-2
II-2
II-2
II-2
II-2
II-2
II-2
II-2
AECA
ColV
Col1, ColV, K>1T
CDC
ELISA
ELISA
No
Nested case control assessed PGD
No
6
ND
ND
ND
ND
j
,
ND
ND
II-2
II-3
II-2
GAD, ICA
GAD, IA-2
RIA
RIA
No
No
ND
ND
ND
ND
,
6
II-2
II-2
GAD, IA-2
RIA
No
ND
ND
6
II-2
33
ABOi; ABO-incompatible; AECA, anti-endothelial/epithelial cell antibody; AMR, antibody-mediated rejection; AT1R, angiotensin II type 1 receptor; DCM, dilated cardiomyopathy; EL, evidence level; CDC, complement-dependent lymphocytotoxicity; Col1, anti-collagen 1; ColV, anti-collagen V; CR, cellular rejection; FC, flow cytometry; GAD, antibodies against glutamic acid decarboxylase; GS, graft survival; HLA-id, HLAidentical; I, islet cell transplantation; IA-2, islet antigen 2 antibodies; IHD, ischemic heart disease; IAK, islet cells after kidney transplantation; ICA, islet cell antibodies; IHD, ischemic heart disease; IK, simultaneous islet and
kidney transplantation; K>1T, anti-K->1 tubulin; MICA, major histocompatibility class I-related chain A; NA, not applicable; ND, not determined; Ped, pediatric; PGD, primary graft dysfunction; RIA, radioimmunoassay;
SDS, sodium dodecyl sulfate polyacrylamide gel; XM, crossmatch.
Tait et al.
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Preexisting non-HLA antibodies in kidney transplantation
Shin (185)
2001
58
NA
Sumitran-Holgersson (186) 2002
139
20
Opelz (187)
2005
4048
803 (PRA 1Y50%), 244 (PRA 950%)
Zou (188)
2007
1910
217
Grandtnerová (189)
2008
2
2
Han (190)
2009
392
62
Breimer (92)
2009
147
35
Ismail (191)
2009
60
40
Montgomery (192)
2009
60
60
Toki (193)
2009
57
57
Reinsmoen (108)
2010
97
32
Narayan (194)
2011
1
1
Kerman (195)
2011
52
NA
Jackson (91)
2011
60
14
Flint (196)
2011
37
37
Chung (197)
2011
14
14
Cox (198)
2011
442
17
Solgi (199)
2012
40
8
Preexisting non-HLA antibodies in heart transplantation
Latif (200)
1995
129
29
Warraich (112)
2000
117
32
Morgun (111)
2004
41
NA
Suarez-Alvarez (201)
2006
31
2
Suarez-Alvarez (202)
2007
44
3
Roche (203)
2008
21
21
Smith (204)
2009
616
69
Smith (205)
2009
491
70
Dipchand (206)
2010
80
35
Preexisting non-HLA antibodies in lung transplantation
Smith (207)
1995
85
27
Iwata (208)
2008
10
NA
Bharat (209)
2010
142
41 total, 33 (Col1), 30 (ColV)
Preexisting non-HLA antibodies in islet cell transplantation
Jaeger (210)
1999 10 IAK, 6 IK, 5 I
6 GAD 2 ICA
Huurman (211)
2000
21
10 (4 GAD+IA-2, 3 GAD alone,
3 IA-2 alone)
Bosi (212)
2001
33 IAK, 3 I
1
Antibody
detected
* 2013 Lippincott Williams & Wilkins
TABLE 7.
34
www.transplantjournal.com
studies (213). There are also reports demonstrating that
antibodies against angiotensin II type 1 receptor (108, 195)
and endothelial cell antigens are associated with kidney
transplant rejection (91).
In heart transplantation, antibodies to cardiac myosin
have been shown to be strongly associated with poor graft
outcome in several studies (111, 112, 200), and in a large
study, the presence of lymphocytotoxic IgM autoantibodies
in the patient’s sera was identified as an independent risk
factor for graft rejection (204). No association was found
between pretransplantation DSA to MICA and function of
heart transplants (205).
In liver transplantation, data on a possible impact of
pretransplantation non-HLA alloantibodies other than ABO
isoagglutinins are not available.
In lung transplantation, antibodies to extracellular matrix proteins expressed on lung tissue, such as collagen I and
V, and a gap junctionYassociated protein K->1 tubulin were
significantly associated with higher rates of primary graft
dysfunction, development of HLA antibodies, and chronic
rejection (208, 209). There is also evidence suggesting that
antibodies against undefined epithelial cell antigens have a
deleterious effect on outcome of lung transplantations (207).
In islet cell transplantation, antibodies to glutamic
acid decarboxylase, islet cells antibodies, and insulin autoantibodies are commonly seen, and there is evidence that
they may correlate with graft outcome either in association
with recurrence of original disease or as part of the rejection
process (210Y212).
ANTIBODY TESTING
POSTTRANSPLANTATION
It is well accepted HLA DSA are associated with allograft rejection and graft failure of transplanted organs.
Considerable experimental and clinical evidence points to a
causal effect of DSA. Whether to anticipate and monitor
donor-directed antibodies depends on the characteristics,
demographics, and risk factors of the patient being transplanted (e.g., primary recipient vs. regraft recipient, male vs.
female, nulliparous vs. multiparous, unsensitized vs. sensitized, and transfused vs. nontransfused). In this section, the
evidence supporting (or refuting) the clinical rationale for
posttransplantation monitoring of solid organ transplant
recipients has been tabulated and annotated. Although there
has been consensus of this committee on how or whether to
proceed in each situation, it was not unanimous. Clearly,
there are inherent reasons to support or refute aggressive
posttransplantation approaches should patients begin to
display DSA that were not originally present or show evidence of graft dysfunction. Part of the controversy on
monitoring derives from the lack of validated therapy once
DSA are detected in a stable patient. Although it is easy to
get caught up in the emotion of posttransplantation monitoring, more evidence-based studies will be essential and
critical to reaching uniform consensus.
Introduction
Standard-of-care monitoring of an allograft recipient
consists of repeated physiologic assessment of allograft function (e.g., serum creatinine, FEV-1, ECHO, C-peptide, liver
enzymes, and bilirubin levels). It is also standard of care to
Transplantation
& Volume 95, Number 1, January 15, 2013
perform a diagnostic biopsy of the allograft when dysfunction
arises that cannot be explained by other causes (e.g., obstruction via renal ultrasound or infection via urine culture).
This is true for all transplant patients regardless of their a priori
risk for rejection based on their pretransplantation assessment.
Currently, two clinical tests are required to make a
diagnosis of AMR in kidney recipients: a serum test to detect
DSA and a biopsy to detect evidence of antibody-mediated
injury in the allograft. For kidney transplantation, the criteria for acute AMR were summarized in the Banff ‘03 report (214), which required both a circulating DSA (to donor
HLA or other endothelial antigens) and histology with C4d+
and morphologic evidence of acute tissue injury (i.e., acute
tubular necrosisYlike minimal inflammation, glomerulitis and
peritubular capillaritis, or arterial-transmural inflammation/
fibrinoid change). For chronic AMR, the histologic features
are TG, multilamination of the basement membranes of the
peritubular capillaries, and transplant arteriopathy. The C4d
and DSA criteria are the same as for acute AMR (215). In
contrast to acute AMR, chronic AMR has a long subclinical
phase measured in months to years. In 2011, the Banff working group recognized (216) that there is growing evidence
for C4d-negative AMR in kidney allografts, particularly in
the late or chronic phase (135, 217). Detection of C4d in the
graft microvasculature has the advantage of high specificity
for DSA and demonstrates complement fixation at the level
of the endothelium, independent of the antigenic target. It has
the major disadvantage of requiring local complement fixation by the antibody. Two markers have been shown to detect
antibody interaction with the microvasculature, increased endothelial gene expression (218), and peritubular capillaritis (135).
Although these are less specific than C4d for circulating DSA,
they may afford increased sensitivity and a Banff working
group is looking at further refinement of the pathologic definition of AMR.
The pathologic, clinical, and serologic definition and
prerequisites for AMR in the heart are quite different and
recently published as an International Society of Heart and
Lung Transplantation consensus (219). An important paradigm shift from this consensus was that a clinical definition
for AMR (cardiac dysfunction and circulating DSA) was no
longer believed to be required due to recent publications
demonstrating that asymptomatic (no cardiac dysfunction)
biopsy-proven AMR is associated with subsequent greater
mortality and greater development of cardiac allograft vasculopathy. Additionally, DSA was not always detected during AMR episodes because the antibody may be adsorbed to
the donor heart or may be to non-HLA antigens (these
limitations also apply to the kidney and other organs). Although AMR occurs in transplanted lungs, livers, pancreas,
islets, and multivisceral organs (164, 168, 170, 220Y226), the
criteria to diagnose such rejection are less well established.
If donor-directed antibodies are identified posttransplantation, the question of what to do with this information becomes paramount. Is there suspicion of graft
dysfunction (e.g., rising creatinine in a renal transplant recipient, dyspnea or arrhythmias in a cardiac allograft recipient,
right upper quadrant and flank tenderness, or elevated liver
function tests in a liver transplant patient)? Because there
now appears to be reasonable evidence of C4d-negative AMR,
what should be done if a C4d test is negative? What if no
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
* 2013 Lippincott Williams & Wilkins
donor-directed HLA antibody is detected but a ‘‘for cause’’
biopsy shows histologic evidence of AMR (presumably nonHLA antibodies or adsorption by the graft)? Should treatment
to eliminate antibodies that cannot be identified still be initiated? If one of the goals for transplant patients is to minimize
immunosuppression, should this be a complete contraindication if DSA are present even if there is no evidence of dysfunction or pathology? Similarly, if DSA are present without
any accompanying symptoms of graft rejection, should treatment to eliminate the antibodies be initiated? These and other
questions have no simple answers because the data needed for
these queries typically do not exist, except for case reports and
anecdotes. As such, there are no generally accepted guidelines
of posttransplantation monitoring for AMR. In the following
sections, evidence-based recommendations to monitor for
AMR in solid organ allograft recipients are provided. Recommendations vary depending on whether a patient is considered
at low, intermediate, or high risk to present with AMR. For
the purposes of this discussion, early posttransplantation was
defined as within the first 6 months, whereas late posttransplantation refers to events after 6 months.
Serum HLA Antibody Testing in the Face of
Allograft Dysfunction
AMR occurs in 10% to 20% of cardiac allograft recipients and correlates with an increased incidence of hemodynamic compromise, rejection, greater incidence of cardiac
allograft vasculopathy, and death (142, 227Y229). However,
not all solid organ allograft recipients are at equal risk for
the development of AMR, at least in the early posttransplantation phase. For example, in renal allograft patients who are
transplanted in the face of immunologic memory (e.g., in the
presence of DSA pretransplantation), there is an increased
incidence of AMR (clinical and subclinical) early posttransplantation in the range of 21% to 55% (127, 130, 230Y232). By
comparison, in the absence of a DSA pretransplantation, the
incidence of early AMR is much lower, which is reported to be
1% to 6% in the first year (127, 130, 230Y232). Moreover, in
patients with cPRA of 80% or more, the evidence that they are
at an increased risk for AMR and graft loss is lacking in the absence of a DSA (16). Therefore, when considering the utility of
serum HLA antibody testing early posttransplantation, one must
take into consideration the risk profile of the patient in question.
Causes of late allograft loss in kidney transplantation
are largely identifiable and primarily immune mediated related to (a) recurrent or de novo autoimmune disease, (b)
AMR with chronic injury (TG and peritubular capillary
basement membrane multilayering), or (c) interstitial fibrosis
and tubular atrophy (IFTA) with or without cell-mediated
rejection or polyomavirus nephropathy (217, 233Y236). As
such, the utility of serum HLA antibody testing may have a
greater likelihood of detecting a DSA with the onset of late
graft dysfunction (i.e., increase in serum creatinine or new
onset proteinuria after 6 months after transplantation). This is
especially true when a patient has been documented to be
nonadherent to immunosuppression (236, 237).
Protocol Biopsy Screening for Antibody-Mediated
Rejection Posttransplantation
For the sake of this discussion, a protocol biopsy is defined as one performed in a stable graft without evidence of
physiologic dysfunction (e.g., proteinuria) or in follow-up to
Tait et al.
35
a posttreatment intervention. The use of protocol biopsies in
cardiac allograft recipients is well accepted (219), but their
role in renal and liver transplant recipients is still debated.
Renal and cardiac patients (and likely liver and lung
patients) transplanted with a DSA, whether immunomodulated
or not pretransplantation, have a higher incidence of AMR early
posttransplantation (see above) compared with patients without
DSA. Moreover, studies performing protocol biopsies in renal
and cardiac patients have found an increased incidence of subclinical AMR. Furthermore, in renal recipients, subclinical TG
can be identified, which is associated with chronic injury and late
graft dysfunction (120, 127, 238, 239).
In addition to performing protocol biopsies to detect
subclinical AMR, protocol biopsies can be conducted after
an AMR event to determine the effectiveness of therapy (i.e.,
persistent C4d+, glomeruli, peritubular capillaries, or vessels) and to identify prognostic indicators of outcome (i.e.,
TG and peritubular capillary basement membrane multilayering) (230, 236, 240, 241). Conversely, in the absence of
a pretransplantation DSA, the likelihood of detecting subclinical AMR early posttransplantation by protocol biopsy
in renal transplant recipients is low (127, 236, 242) but may
be more readily apparent in hearts (219).
Recently, Wiebe et al. (236) performed ‘‘protocol biopsies’’ in patients in whom the only abnormality was a de novo
DSA in the serum; whether these should be considered ‘‘protocol’’ or ‘‘for cause’’ is a matter for debate. Nonetheless, this
group demonstrated that 10 of 14 biopsies in stable kidney
transplant patients with a de novo DSA had evidence of
microvascular inflammation (glomeruli, peritubular capillaries, and vessels with or without C4d+) consistent with subclinical AMR.
The significance of de novo HLA antibody in the absence of de novo DSA HLA antibody is debatable. Groups
have reported excellent outcomes in patients with pretransplantation or de novo HLA antibody (16, 236, 243, 244),
whereas others have suggested an increased risk for graft loss,
citing the DSA as potentially being sequestered in the allograft
preventing its detection in the circulation (244Y251). What is
often debated is the significance of de novo HLA antibodies
that are not donor directed. Lacking is a study whereby a
protocol biopsy was initiated based on a de novo HLA antibody without a DSA being present to document whether any
evidence of antibody-mediated injury is present. It is notable
in the serial study by Wiebe et al. (236) that only 9% who
developed de novo DSA had an earlier serum where only de
novo HLA antibody was detectable, suggesting that de novo
HLA antibody is generally not a marker of de novo DSA in the
kidney allograft.
Experience with the use of protocol biopsies to study
patients undergoing immunosuppression withdrawal is
limited. However, a recent study demonstrated that patients
who were switched from cyclosporine to everolimus at 3 or
4.5 months after transplantation had a higher rate of de
novo DSA and AMR after cyclosporine withdrawal compared
with patients maintained on cyclosporine (252). Similarly,
nonadherence, a form of immunosuppressive withdrawal, is
associated with a higher rate of de novo DSA and accelerated
graft loss associated with AMR (236, 237). Taken together,
it would suggest that the reduction of immunosuppression
could carry an increased risk for AMR.
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36
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Serial Serum HLA Antibody Screening
Posttransplantation
Kidney patients transplanted in the face of a DSA pretransplantation have an increased incidence of AMR early
posttransplantation as well as subclinical AMR and TG (see
above). Several groups have demonstrated in such patients that
the persistence of, or an increase in, DSA in the serum correlates with a poor graft outcome regardless of whether the early
graft function is stable or not (120, 216, 230, 253, 254). Similarly, failure of the DSA to decrease posttreatment for AMR has
been associated with a poor graft outcome (253, 255, 256).
The development of de novo DSA after renal transplantation has been associated with increased risk for graft
loss in many large series (35, 236, 243Y247, 257Y259). Of
note, in many of these reports, although de novo class I DSA
occurs, most patients develop de novo class II DSA directed
at donor DR, DQ, and, occasionally, DP antigens.
A recent study followed the evolution of de novo DSA
and demonstrated that the mean time to appearance is 4.6
years after transplantation with a tendency to appear sooner
in the face of nonadherence (236); the prevalence of de novo
DSA at 10 years was 20% in adherent patients and 60% in
nonadherent patients. A similar high prevalence of noncompliance in patients with chronic AMR was recently
reported (237). In addition to drug minimization or nonadherence, other risk factors for the development of de novo
DSA are HLA-DR mismatching, early cell-mediated rejection (both clinical and subclinical), and younger recipient
ages (236, 245, 252, 260).
With regard to cardiac transplantation, the deleterious
effect of alloantibodies on graft survival, particularly in patients
who developed de novo alloantibodies posttransplantation, was
recently reported. In a group of 950 patients studied over a
15-year period, graft survival was highest in patients who never
developed alloantibodies (70%) or who displayed them only
pretransplantation (71%); graft survival was lower in recipients who showed antibodies both pretransplantation and
posttransplantation (56%) or only posttransplantation (47%).
De novo antibodies appearing more than 1 year after transplantation had the poorest survival. The development of AMR
had a significant negative impact on graft survival (16% in
AMR vs. 63% in no AMR patients) (261).
Among lung transplant recipients, the presence of
HLA antibodies has been associated with persistent recurrent acute rejection (220). Importantly, rejection is the most
significant risk factor for the development of bronchiolitis
obliterans, which has an incidence as high as 50% posttransplantation (223Y225) and is associated with the development of DSA (221). Whether treatment to eliminate/
reduce DSA mitigates the risk of lung rejection is still unclear
(221). In liver allograft recipients, although HLA antibodies
have recently emerged as contributing factors to episodes of
acute and chronic rejection (164, 168, 170), whether antibody
depletion has any beneficial effect has not yet been reported.
This is also true for recipients of other organ transplants.
Other Considerations
Should Non-HLA Antibodies Be Considered for
Posttransplantation Assessment?
The question of whether other non-HLA DSA should
be considered posttransplantation is one of growing interest.
Transplantation
& Volume 95, Number 1, January 15, 2013
AMR has been reported, rarely, in HLA-identical sibling
renal allografts, presumably due to non-HLA antigenic targets (262, 263). There have been a number of reports of
MICA antibodies associated with poor graft survival (188,
264, 265). The prevalence of MICA antibodies after kidney
transplantation was 8.9% versus 26.8% for HLA antibodies
in one series (264) and MICA antibodies were present pretransplantation in 11.4% of patients in another series (188).
The issue with these articles is that donor specificity is not
proven and correlation with a pathologic outcome was absent. Therefore, although the data support the association
with allograft failure, the hypothesis that MICA antibodies
are causal has not been proven.
Dragun et al. (107) reported angiotensin II type 1 receptor activating IgG antibodies in renal allograft rejection.
However, it should be noted that these patients tended to have
a unique presentation with malignant hypertension, graft
dysfunction, and histology showing endarteritis and fibrinoid
necrosis that occurred at a median of 4 days after transplantation (range, 2Y1217).
AECA of undefined specificity must also be considered
as potential mediators of graft rejection. Recent studies have
suggested that preexisting and de novo AECA are associated
with a risk of early graft rejection (74, 87, 90, 91). Similar to
the MICA antibodies described above, the donor specificity of
these antibodies has not been conclusively proven.
Recently, groups using protein microarray technology
have been reporting the association of autoantibodies (e.g.,
anti-peroxisomal-trans-2-enoyl-coA-reductase) with the development of TG or IFTA in kidney allografts (95, 97). Whether
any of the above antibodies are the cause or consequence of the
pathogenesis of TG and IFTA has yet to be determined.
Consensus Recommendations
Technical Group
1) Antibody identification
a. At least one SPI should be used to detect and characterize
HLA class I and IIYspecific antibodies. A SAB immunoassay should be performed at least once pretransplantation in HLA-immunized patients. This is particularly
important for the characterization of antibodies directed
at Cw, DQA, DPA, and DPB, which are not adequately
defined by other techniques. [1]
b. Use both SPI and cell-based assays to assess antibody
status to the intended donor. [1]
c. Laboratories must correlate the level of antibody detected
by SPI with cell-based assays to establish the likelihood of
a positive XM. [1]
2) Standards for cell-based assays (CDC)
a. CDC assays for antibody identification and cross-matching
should be performed using target cells that permit identification of antibodies to both HLA class I and II antigens. [1]
b. Nonspecific reactivity must be recognized. [1]
c. Consider modifications to increase sensitivity and specificity including wash steps, changes in incubation times,
addition of antiglobulin, and serum modification steps to
remove or inactivate IgM and C1. [2]
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tait et al.
* 2013 Lippincott Williams & Wilkins
3) Standards for flow cytometry cell-based assays (flow
cytometry)
a. Differentiation of T and B cells should be performed by
a three-color fluorescence technique. [1]
b. Consider modifications such as Pronase use to increase
sensitivity and specificity. [3]
4) Standards of practice
a. The laboratory performing tests on transplant patients
must have documented expertise in antibody assessment
and interpretation. [1]
b. Each laboratory must establish its own threshold for antibody specificity assignment and clinical interpretation. [1]
c. Each center should define changes in MFI values between sera from the same patient that are clinically
meaningful. [2]
d. The patient history must be considered for the interpretation of antibody screening and interpretation of test
results. Factors include the history of parity in female
patients and previous graft HLA mismatches. Such information indicating a possible state of presensitization
despite low levels of antibodies can put greater clinical
emphasis on low-level antibodies than would normally
occur. Consideration of prior immunologic history can
also assist in the recognition of naturally occurring antibodies to denatured HLA antigens in patients who have no
obvious cause of sensitization. [1]
e. In determining antibody specificity, the laboratory should
consider the possibility of antibodies to epitopes on any
polymorphic chains (including DQA and DPA) as well as
epitopes resulting from combinations of different > and
A chains. [2]
f. HLA typing of donor and recipient must be performed at a
level required for accurate antibody interpretation. [1]
g. Store donor material in the form of frozen cells and DNA
for posttransplantation DSA investigations. [1]
5) Interfering factors in interpretation of SPI
a. Consideration must be given to the following variables
when performing and assessing HLA antibody results:
antigen density on beads and condition (i.e., denatured
Ag); reactivity of control sera and control beads; reduction
of test interference (i.e., EDTA, DTT, and hypotonic dialysis); and when saturation of target antigens may have
occurred, sera should be tested under conditions where
meaningful changes in antibody levels can be detected
(e.g., serum dilutions). [1]
6) Assay standardization
a. Laboratories should follow standardized operating procedures and policies that minimize test variability including,
wherever possible, robotic processing, temperature control,
consistency in washing procedures, and instrument calibration. [1]
b. Quality-control procedures must be introduced to monitor interassay and intraassay variability. [1]
c. Each laboratory must participate in relevant external proficiency testing programs as required by local, regional, and
national regulations. [1]
7) Reporting of results
a. The following points should be included in the reporting
format:
1. Sample and assay dates
2. Assay name
37
3. Calculated reaction frequency/cPRA/virtual PRA indicates
the frequency of donors with unacceptable HLA antigen
mismatches.
4. Specificity assignment and assessment of antibody level.
Note that SPI have not been approved for reporting of
quantitative measurements. As such, MFI values do not
necessarily reflect antibody titer.
5. Comments on presence/absence of DSA if a specific
donor is being assessed
6. Immunoglobulin class and isotype if available
7. Assay or serum modification employed. [3]
Pretransplantation Group
8) Transplantation risk stratification categories should be developed based on antibody identification and XM results. [3]
9) Information regarding prior sensitizing events should be
considered in interpreting antibody testing results. [2]
10) DSA detected by CDC antibody screening and crossmatching in the most recent serum collected must be
avoided because they are associated with a high risk for
AMR and graft loss. [1]
11) To minimize risk of sensitization and antibody-mediated
allograft damage, administration of blood products
pretransplantation should be avoided if possible. [1]
12) When a patient is sensitized, precise characterization of
HLA antibodies and complete HLA typing of the donor
pretransplantation must be performed. [1]
13) HLA antibody screening should be performed at a frequency that accommodates the likelihood of an imminent transplantation based on local waiting times and
the immunologic risk of adverse outcome such as in
highly sensitized patients. [3]
14) A minimum of two sera obtained at different time points
should be tested to confirm presence or absence of HLA
antibodies. [3]
15) Sera should be tested after known sensitizing events,
proinflammatory events, and at regular intervals once
listed for transplantation. [1]
16) Kidney
a. Unacceptable HLA antigens should be a part of kidney
allocation algorithms. [2]
b. Accurate XM prediction depends on complete HLA typing.
To minimize the incidence of unexpected positive XM in
paired exchange registries, the donor should be typed at
HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLADRB4, HLA-DRB5, HLA-DQA, HLA-DQB, HLA-DPA,
and HLA-DPB loci. [2]
c. A renal transplantation can be performed without a
prospective pretransplantation CDC or flow XM if SAB
testing indicates the consistent absence of DSA against
HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLADRB4, HLA-DRB5, HLA-DQA, HLA-DQB, HLA-DPA,
and HLA-DPB locus antigens. Each center needs to develop its policy in agreement with regulatory bodies and
clinical programs. [3]
d. Risk assessment should include HLA antibody specificities identified in historic sera. [3]
e. In renal transplantation, if DSA is present but the CDC
XM against donor T and B cells is negative, this should
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38
www.transplantjournal.com
be regarded as an increased immunologic risk but not
necessarily a contraindication to transplantation, especially
after elimination of DSA by desensitization. [2]
f. To optimize access to transplantation of highly sensitized
recipients, inclusion of patients in special programs, such
as kidney paired donation, AM, or desensitization, should
be considered. [1]
g. HLA matching should be part of the allocation procedures
to reduce the probability of developing HLA antibody,
rejection, and graft loss. [2]
h. ABO incompatibility is no longer an absolute contraindication in kidney transplantation and ABO-incompatible
transplants can be successfully performed in recipients in
whom isoagglutinin titers have been lowered to acceptable
levels. [1]
i. Based on current evidence, no recommendation can be
made for routine pretransplantation testing for nonHLA antibodies other than ABO. [2]
17) Heart
a. In both pediatric and adult heart transplantation, determination of pretransplantation DSA must be performed
because it is critical to improve short-term outcomes and
preventing early acute rejection. [1]
b. Desensitization therapy should be considered in sensitized
heart transplant recipients. [2]
18) Lung
a. Pretransplantation DSA in recent serum should be
avoided in lung transplantation whenever possible. [1]
19) Liver
a. The liver allograft may be partially resistant to antibodymediated damage; however, high-level DSA antibody may
be associated with inferior outcomes and should be considered as a risk factor for graft dysfunction. [2]
b. Pretransplantation screening for HLA antibodies is
recommended in liver transplant recipients for risk stratification. [3]
c. Donor tissue should be collected and stored in liver
transplantation. [3]
d. An XM should be performed in sensitized liver transplant
recipients. [2]
e. In sensitized recipients of combined liver-kidney transplantation, the liver may not confer full protection for
preventing AMR in the kidney and should be included in
risk assessment. [2]
20) Pancreas
a. Recommendations for kidney transplantation should
apply to the pancreas for simultaneous pancreas-kidney
transplantation. [1]
b. Pancreas is at risk for AMR and pretransplantation DSA
should be avoided whenever possible. [1]
c. In pancreas transplantation, AMR should be considered
in the differential diagnosis of early graft thrombosis and
graft dysfunction. [2]
21) Intestine
a. In intestinal transplantation, pretransplantation HLA
antibodies should be determined. The risk assessment
should be based on the level of DSA. [2]
22) Islets
a. Based on the available literature, pretransplantation DSA
are associated with impaired islet cells function posttransplantation and should be avoided. [2]
Transplantation
& Volume 95, Number 1, January 15, 2013
Posttransplantation Group
23) Pretransplantation
a. DNA must be available on all donors for identification of
donor antigens. This is essential for accurate DSA assessment. [1]
b. Store frozen pretransplantation serum from recipients
(acceptable is j20-C; recommended is j80-C). The most
current serum is acceptable; day of transplant serum
recommended. [1]
24) Posttransplantation (months 0Y12)
a. Very high risk patients (desensitized): These patients
are recognized to be at high risk for early clinical or
subclinical AMR and as such are treated with a desensitization protocol. Such protocols are not standardized
and are center specific. Monitor DSA and conduct
protocol biopsies in the first 3 months after transplantation. [1]
1. If there is evidence of clinical or subclinical AMR, the
patient should be treated. Efficacy of treatment is reflected as normal graft function and is associated with
a reduction of DSA levels (253, 266). [2]
2. If there is a rapidly increasing level of DSA accompanied by a biopsy showing no rejection, initiation of therapy to reduce the DSA levels should be considered. [3]
b. High-risk patients (DSA positive/XM negative): These
patients are recognized to be at risk for early clinical or
subclinical AMR. Monitor DSA and conduct a protocol
biopsy in the first 3 months after transplantation. [1]
1. If biopsy is positive for AMR, the objective is to treat.
Efficacy of treatment is reflected as normal graft
function and is associated with a reduction of DSA
levels (253, 266). [2]
2. If there is a rapidly increasing level of DSA accompanied
by a biopsy showing no rejection, initiation of therapy to
reduce the DSA levels should be considered. [3]
3. DSA persists in the absence of biopsy proven rejection,
immunosuppression should not be reduced and additional monitoring should be considered. [3]
4. If the DSA and biopsy are negative, follow as if low
risk (see d.1.) unless there is an inflammatory event,
in which case additional monitoring for DSA is recommended. [2]
c. Intermediate-risk patients: Includes history of sensitization to donor antigen(s) by CDC and SPI but currently
negative and history of sensitization with at least one
positive test for HLA antibodies. Monitor for DSA
within the first month. [2]
1. If a DSA present, then perform a biopsy. A biopsy is
recommended because of published data that document an association between DSA and clinical or subclinical rejection. [2]
2. If biopsy is positive for rejection, the objective is to
treat. Efficacy of treatment is reflected as normal graft
function and is associated with a reduction of DSA
levels (253, 266). [2]
3. In the absence of biopsy-proven rejection, additional DSA
monitoring should be considered within the first year. [3]
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
* 2013 Lippincott Williams & Wilkins
4. Patients with a DSA in the absence of biopsy-proven
rejection should not be considered for reduction in
immunosuppression. [3]
5. In the absence of a DSA follow-up as if low risk (see
d.1.). [2]
d. Low-risk patients (nonsensitized, first transplantation)
1. Screen for DSA under the following circumstances:
a) at least once 3 to 12 months after transplantation. [2]
b) whenever significant change in maintenance immunosuppression is considered (e.g., minimization/withdrawal/
conversion). [2]
c) suspected nonadherence. [2]
d) graft dysfunction. [2]
e) before transfer of care to a remote center outside the
transplant center. [3]
2. If DSA present, then perform a biopsy. A biopsy is recommended because of published data that document
an association between DSA and clinical or subclinical
rejection. [2]
3. If the biopsy is positive for rejection the objective is to
treat. Efficacy of treatment is reflected as normal graft
function and is associated with a reduction of DSA levels
(253, 266). [2]
4. In the absence of biopsy-proven rejection additional
DSA, monitoring should be considered within the first
year. [3]
5. Patients with a DSA in the absence of biopsy-proven
rejection should not be considered for reduction in
immunosuppression. [3]
6. If no DSA present, then no additional testing in the first
year is recommended in the absence of circumstances
listed under point 1 above. [2]
25) Posttransplantation (month 12 onward)Vapplies to all
risk categories
a. Store at least one serum sample per year (i.e., on the
transplantation anniversary). [3]
b. Evaluate DSA in a current serum if any of the following
conditions occur:
1. Significant change in maintenance immunosuppression is considered (e.g., minimization/withdrawal/
conversion). [2]
2. Suspected nonadherence. [2]
3. Graft dysfunction. [2]
4. Before transfer of care to a remote center outside the
transplant center. [3]
c. If de novo DSA present or if there is an increase in
previous DSA levels, perform a biopsy. A biopsy is recommended because of published data that document
an association between DSA and clinical or subclinical
rejection. [2]
1. If biopsy is positive for AMR, the objective is to treat.
Efficacy of treatment is reflected as normal graft
function and is associated with a reduction of DSA
levels (253, 266). [2]
2. If biopsy is negative (no sign of rejection) monitor
the DSA and monitor for a change in graft function. [3]
3. Patients with a DSA even without biopsy proven rejection should not be considered for reduction in immunosuppression. [3]
Tait et al.
39
Note regarding low-risk patients: The recommendations of posttransplantation monitoring beyond the first
year represent the majority opinion of the group. There was,
however, a minority dissenting voice that supported at least
annual testing on the collected sample(s). The rationale of
the minority was that early detection of DSA would allow
the clinician to optimize patient care (e.g., perform a biopsy
and avoid immunosuppressive minimization). The majority
voice focused on the high cost of additional testing on all
patients when the anticipated incidence of de novo DSA is
less than 5% per year coupled with the lack of proven effectiveness of early intervention for late AMR (267).
Additionally, the aforementioned recommendations
refer to noncardiac transplantation monitoring. In the case
of low-risk heart transplant patients, the reader is referred to
the recently published consensus article in the Journal of
Heart and Lung Transplantation (219).
Recommendation grades are shown in brackets at the
end of each statement. (See Introduction for explanation of
grades.) The grades are based on those outlined by Uhlig K,
McLeod A, Craig J et al. Grading evidence and recommendations for clinical practice guidelines in nephrology: a position statement from Kidney Disease: Improving Global
Outcomes (KDIGO). Kidney Int 2006;70:2058.
Future Directions and Research
Technical Group
1. A coordinated search for a selected bank of HLA reference antibodies to be used for assessing the interlaboratory variability in SPI antibody testing should be
undertaken. Antibodies to MICA and relevant non-HLA
antigens should be included.
2. The C1q modified single-antigen antibody identification
requires more research and validation to understand its
application to risk assessment and monitoring efficacy of
treatment.
3. If production of the SPI donor-specific XM kit is continued, further research will be required to establish the
clinical role of such a test. Specifically, the sensitivity of
the XM in relation to SAB testing requires further
investigation.
4. The clinical endothelial cell XM for the detection of antiendothelial antibodies needs further investigation.
5. SPI for the detection of non-HLA anti-endothelial
antibodies require development.
6. Multicenter studies are required to establish the clinical
utility of testing for antibodies to tissue-specific nonHLA antigens such as the angiotensin II type 1 receptor.
Further development of SPI for the detection of these
antibodies is warranted based on initial findings.
7. The role of antibodies to epitopes found on HLA-E
merits further investigation.
8. The short-term and long-term clinical effect of low levels
of HLA antibodies detected by SPI requires further
investigation.
Pretransplantation Group
9. More systematic studies for immunologic risk assessment
that include long-term outcome data are required.
10. Randomized controlled studies to analyze the efficacy and
safety of different desensitization protocols are required.
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40
www.transplantjournal.com
11. Additional studies are needed to determine the role of
preexisting complement fixing versus noncomplement
fixing antibodies to HLA and their role in organ transplantation.
12. Efficacy of desensitization in achieving good long-term graft
survival needs to be established in heart transplantation.
13. Efficacy of desensitization in achieving good long-term graft
survival needs to be established in lung transplantation.
14. More systematic studies are required on the impact of
DSA on pancreas transplant alone or in combination
with a kidney.
15. Detailed analysis is required to establish the impact of
pretransplantation DSA on outcomes in intestine and
multivisceral transplantation.
16. Further studies are needed to determine the risk of preexisting DSA in islet cell transplantation.
17. Standardization of the methodology for determining
ABO isoagglutinin titers is required.
18. Future studies are required to define the role of preexisting
antibodies to non-HLA and self- antigen on the outcome of
solid organ and cellular transplantations.
Posttransplantation Group
A need is recognized for the following:
19. Serial screening of serum to determine timing of onset of
de novo DSA before onset of graft dysfunction.
20. Protocol biopsies at first appearance of de novo DSA to
document pathologic correlation.
21. Assessment of DSA for complement fixing activity and
correlation with clinical events (e.g., DSA C1q binding
and IgG subclass specificity of DSA).
22. Clinical trials that include serial DSA monitoring posttreatment and posttreatment biopsies to correlate DSA
levels with histologic response to therapy and long-term
outcome.
23. Clinical trials to prevent production of DSA.
Future Directions
Although DSA are routinely found in patients who
experience immunologic graft loss, whether the antibodies
are causal or a consequence of other process(es) was, at least
until recently, an unanswered question. As such, it has been
a challenge to know what, if anything to do when antibodies
are identified posttransplantation. Clearly, reemergence of
DSA that were present immediately pretransplantation,
which required the recipient to be desensitized in order for
the patient to be transplanted, requires an immediate response to try and prevent a catastrophic event.
The response of any given patient to therapy can span
a huge spectrum, ranging from complete elimination of
DSA and the accompanying symptoms of graft rejection to a
failure to eliminate or even reduce the DSA while the patient
steadily proceeds to graft rejection. Studies over the past
several years have indicated that certain patients (e.g., those
with pretransplantation CDC titers of DSA 91:16) (268) do
extremely poorly after transplantation even when the antibodies appear to be completely eliminated pretransplantation after desensitization and therefore should not be entered
into a desensitization program. Thirty percent of patients who
undergo desensitization still experience AMR and are at risk
to develop TG and ultimately graft loss (269). To date, there is
Transplantation
& Volume 95, Number 1, January 15, 2013
no predictive test to identify into which category (the 70%
of patients who do well or the 30% who experience AMR)
the patient will fall (269). Newly developed tests aimed at
detecting and quantifying memory B cells (270) and plasma
cells (271) that produce DSA may eventually be used to help
categorize a patient’s posttransplantation risk to have transplant
threatening DSA reappear. But until those tests become reliable, the risk of any given desensitized patient cannot be accurately assessed. So, too, is the risk of any posttransplantation
patient to develop DSA. Collective experience indicates that
15% to 20% of patients will develop de novo DSA posttransplantation (236, 272). How frequently posttransplantation
candidates without pretransplantation DSA should be monitored and what to do if antibodies are identified are ‘‘hot button’’ questions.
Experimental studies undeniably document the pathologic role of DSA in allograft rejection in animal models.
One of the most convincing studies was described by Russell
et al. (273) who used a severe combined immunodeficient
mouse model of heart transplantation where cellular rejection was obviated. They observed that the passive transfer
of alloantisera containing antibodies to donor histocompatibility antigens led to the development of obstructive
coronary lesions, the equivalent of transplant coronary
artery disease in humans. The result of this study cannot be
extrapolated to humans and although the data supporting
the humoral theory of rejection are indirect and subtler,
the collective data have become quite compelling (reviewed
in (272) particularly when using SPI approaches to identify donor-directed HLA antibodies). Specifically, DSA
are found in most of the kidney (249, 258, 274), heart (65,
275), lung (275), islet (226), and liver (164, 168) transplant
patients who have rejected their grafts, and most studies
demonstrate that the appearance of DSA precedes graft
loss, lending credence to the tenet that these antibodies, although not necessarily causal of rejection, are not a consequence of rejection. Although recent studies have examined
the effect of antibody depletion (with bortezomib and
combination therapy with plasmapheresis/IVIg and rituximab) and have shown to curtail the symptoms of AMR
(266), the long-term benefit of such treatment has not
been documented.
A recent FDA workshop that focused on the treatment
and prevention of AMR raised a number of concerns about
how data are generated, interpreted, and used (267). The
need to standardize and refine antibody detection assays was
recognized to be paramount. Although such tests are currently being used to define DSA (and, by extension, diagnose AMR), they are far from perfect. Although there is a
desire to use these tests to quantify antibodies and to
monitor the effectiveness of therapy, it was noted that these
assays have not been validated or approved for quantitative
assessment (267). Finally, emerging (although controversial)
data suggest that the analysis of antibody function (e.g.,
complement fixation) may be a useful indicator of AMR risk
(71, 276).
Determining clear surrogate endpoints for effective
AMR treatment has also been called for by the FDA; whether
antibody levels can serve this purpose is as yet unclear.
Compounding the problems is the lack of an effective
treatment option for late-developing AMR (241). Can early
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Tait et al.
* 2013 Lippincott Williams & Wilkins
intervention prevent graft loss due to AMR? Answering that
question will ultimately require multicenter randomized
control trials, which is exactly what the participants in the
FDA workshop on AMR concluded. It is only with such
foresight that effective strategies can be developed to treat,
reverse, and prevent AMR.
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Appendix
The composition of the three working
groups who contributed to this consensus
report is as follows:
1. Technical Group:
Chairpersons: Brian D. Tait (Australia) and Andrea A.
Zachary (USA).
Robert A. Bray (USA), Ilias I.N. Doxiadis (The Netherlands), Nils Lachmann (Germany), Elaine F. Reed
(USA), Craig J. Taylor (UK), and Dolly B. Tyan (USA).
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* 2013 Lippincott Williams & Wilkins
2. Pretransplantation Group
Chairperson: Caner Süsal (Germany).
Patricia Campbell (Canada), Jeremy R. Chapman (Australia),
Angela Webster (Australia), Frans H.J. Claas (the
Netherlands), Susan V. Fuggle (UK), Suchitra SumitranHolgersson (Sweden), Thalachallour Mohanakumar
(USA), and Adriana Zeevi (USA).
Tait et al.
47
3. Posttransplantation Group
Chairpersons: Peter W. Nickerson (Canada) and Howard
M. Gebel (USA).
P. Toby Coates (Australia), Robert B. Colvin (USA),
Emanuele Cozzi (Italy), John Gill (Canada), Denis Glotz
(France), Nicole Suciu-Foca (USA), and Kazunari
Tanabe (Japan).
Copyright © 2012 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.