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Summary. Transplant recipients who have undergone
sensitizing events, such as pregnancy, blood transfusion
or previous transplants, frequently develop antibodies
di r ecte d ag a inst the hig h ly p olym o rphou s hu man
leukocyte antigen (HLA)-molecules. These pre-formed,
donor-specific antibodies (DSA) present a high risk of
causin g organ failure or even complete loss of the
grafted organ as a consequence of antibody-mediated,
hyper-acute or acute allograft rejection. In order to
detect DSA, the so-c alled functional complem ent-
dependent lymphocytotoxicity assay (CDC-XM) was
established about 50 years ago. Although effective in
improving the outcome of solid organ allo-grafting, for
the last ten years this assay has been controversially
discussed due to its low sensitivity and especially
because of its high susceptibility to various artificial
factors, which generally do not yield reliable results. As
a consequence, novel immunochemical test systems
have been developed using ELISA- or bead-based solid
phase assays as replacements for the traditional CDC-
based assays. Because these assays are independent of
single or vital cells, which are frequently not available,
th e y ha v e p r ovided an addit i onal and alter n ative
diag nostic approach compare d wit h the trad itional
CDC-ba s e d and flow-cytomet r i c analys e s .
Unfortunately, however, the AMS-ELISA (Antibody
Monitoring System), which was the first system to
become commer c i a l l y av a i l a b l e , wa s re c e n t l y
discontinued by the manufacturer after seven years of
successful use. Alternative procedures, such as the
AbCros s - E L I S A , had to be either co n s i d e r a b l y
modified, or did not yield reliable results, as in the case
of the Luminex-based assay termed DSA. We draw the
conclusion that due to the unique features and fields of
application reviewed here, the implementation of solid
phase cross-matc h i n g still represent s an urgent
requirement for any HLA-laboratory’s routine tasks.
Ke y w o rds: A llog raft, Cr ossm atch (X M), Don or-
specific antibodies (DSA), Human leukocyte antigen
(HLA), Rejection
Ben e f i t s and drawb a c k s o f the conve n t i o nal
crossmatch procedures: Complement-dependent
ly mph ocytoto xic ity (C DC-) a nd flo w-cy tom etr ic
crossmatch assays
It has been known for about 50 years that antibodies
which are directed against antigens of donor tissue
represent the main reason for hyper-acute and acute
rejections of renal allografts, as well as of other organ
all o g r afts ( P a tel a n d Tera s a k i , 19 6 9 ) . Nu m e r ous
subsequent studies have shown that these deleterious
antibodies were primarily directed against antigens of
the human major histocompatibility complex (MHC),
the so-called human leukocyte antigens (HLA) (Ahern et
al., 1982; Chapman et al., 1986). These donor-specific
anti -HLA antibodies (DSA) a re thus rega rded as a
con t r a -indicat i o n fo r gr afting as defi n e d by th e
Review
Solid phase-based cross-matching for solid
organ transplantation: Currently out-of-stock but
urgently required for improved allograft outcome
Gerald Schlaf1, Daniela Bau1, Nathalie Horstmann1, Gary Sawers2and Wolfgang Altermann1
1Tissue Typing Laboratory (GHATT), University Hospital and 2Institute of
Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Halle/Saale, Germany
Histol Histopathol (2020) 35: 937-948
http://www.hh.um.es
Offprint requests to: Gerald Schlaf, Transfusion Medicine, Tissue Typing
Laboratory (GHATT), University Hospital, Martin-Luther University Halle-
Wittenberg, Ernst-Grube-Straße 20, D-06120 Halle/Saale, Germany. e-
mail: gerald.schlaf@uk-halle.de
DOI: 10.14670/HH-18-217
istology and
istopathology
From Cell Biology to Tissue Engineering
H
tran s p lantati o n gu i d e lines of m o st c o untries and
supranational societies (e.g. Eurotransplant Foundation),
which supervise the allocation of kidneys and other solid
organs.
The so-called crossmatch (XM-) procedure was
developed in the late 1960s to detect antibodies in a
given recipient’s serum that reacted with lymphocytes
isolated from the donor’s blood (Patel and Terasaki,
1969). Even today, a negative crossmatch outcome in
such a test is regarded as the best predictor for short-
term survival of renal allografts. The standard technique
established originally was the complement-dependent
lymphocytotoxicity (CDC) assay and the work-flow of
this assay has b een descr ibe d i n detail previousl y
(Altermann et al., 2006). Using this functional vitality
assay, only those DSA were detectable that exert their
detrimental function by their complement-activating
fe a tures , ul timate ly l eadin g to the lys is o f do nor
lymphocytes. Additional DSA including isotypes other
than IgM, IgG1 and IgG3 without complement-fixing
activity are not detectable by this method, although these
can be just as detrimental for donor tissues/organs. The
second drawback of this assay is its low sensitivity
lea d i n g to its g e neral in a b ility to d e tect lo w
concentrations of DSA. Consequently, the CDC was
mod i f i ed b y intro d u c i ng s e condary anti-huma n
immunoglobulin (Ig) antibodies, which recognize the
pr imary DSA, there by supp lem enti ng this a ssay ’s
technical design in order to enhance its complement-
activating potency (Gebel and Bray, 2000; Karpinski et
al., 2001; Altermann et al., 2006). The procedure was
termed anti-human globulin- (AHG-) enhanced CDC-
XM. The additional incubation time required in this
assay was a “stress factor” that sometimes could lead to
a higher background and possibly to uninterpretable
results due to a high rate of artificially induced cell
de ath. Th is was esp ecia lly a prob lem whe n u sing
donors’ cells pre-damaged during inadequate storage
conditions and extended storage times prior to the CDC-
XM.
In order t o c irc umvent so me of the C DC-XM-
specific problems t he flo w-c yto met ric cr oss mat ch
(FAC S - X M) wa s establ i s h ed, w h i c h all o w e d the
detection of complement-activating as well as
compl eme nt- ind ependent DSA (Lo bo et al., 1981;
Ga rovoy et al . , 1 983; Sco rnik et al. , 1 9 94, 199 7 ;
Bi ttenc ourt et al., 19 9 8; Chris tiaan s e t al ., 1 998;
Karpinski et al., 2001; Altermann et a l., 2006). Its
sensitivity is in the range of the AHG-enhanced CDC-
crossmatch. Frequently, controversial results have been
published concerning the significance of FACS-based
cross-matching. For example, it was demonstrated that a
positive outcome does not necessarily correlate with an
increased number of rejection episodes (Kerman et al.,
1999; Lobashevsky et al., 2000) or it was also shown
tha t all o a n tibodies , whi c h do not activa t e th e
complement system, were actually associated with an
increased number of rejections, despite the
corre spo nding CDC-XM results showing negative
outcomes (Scornik et al., 1994; Scornik, 1995). These
discrepancies, however, were most probably due to a
pivotal problem regarding signal-specificity, which, even
today is still a problem with this assay. In particular, the
FACS-XM of isolated B-cells is frequently influenced by
artefacts due to the “irrelevant/unspecific” binding of
antibodies through their Fc-domains to the Fc-receptors
expressed on this cell type ultimately leading to false
posi tive results. In con trast to T-cells to which n o
bin d i n g of DSA was demo n s t rable, ou r o w n
investigations have shown that the B-cell crossmatch is
often characterized by a positive signal due to a shift in
the FACS-histogram (Altermann et al., 2006). It is
noteworthy, however, that most of these positive flow-
cytometric B-cell crossmatch outcomes were neither
confirmed virtually, i.e. by identifying the respective
antibodies through the use of corresponding anti-HLA
class II screen and specification assays, nor were they
confirmed by conventional CDC-cross-matching using
isolated B-cells, suggesting that the problem represents a
common rather than a rare event (Altermann et al., 2006;
Delgado and Eckels, 2008).
The approach to pre-digest lymphocytes with the
en z y me-mi x ture prona s e in ord e r to inc r ease the
specificity of DSA binding was introduced by some
groups (Lobo et al., 1995; Vaidya et al., 2001a) but the
specificity-enhancing effects were not shown to be
reproducible by several other groups, including our own
laboratory, and did not perform as well as the FACS-
based cross-matching approach. The theoretical benefit
of using pronase was doubtful from the beginning, as this
is not a single protease with defined proteolytic cleavage
sites, such as pepsin, but a complex mixture of non-
specific bacterial proteases, which is isolated from the
extracellular fluid of Streptomyces griseus. Its activity
extends to both denatured and native proteins leading to
almost complete digestion to the single amino acid level.
Due to this lack of specificity, the general reproducibility
of this procedure was strongly challenged, particularly as
a conseque nce of the pronase-mediate d loss of Fc-
receptors and of HLA-molecules. Indeed, the findings
indicated that Fc-receptors are not the primary target of
pronase treatment, as was recently summarized (Brown et
al., 2017). Furthermore, Fc-receptors and HLA molecules
both belong to the immunoglobulin superfamily, which
indicates structural homologies. This was contrary to the
claims of initial publications, all of which originated
from two groups (Lobo et al., 1995, 1997, 2002; Vaidya
et al., 2001a,b; Bearden et al., 2004) and highlighted the
benefit of using pronase. Meanwhile, nearly all of the
mo r e r ecent l y p ublis h ed studi e s h ave cle a r ly
demonstrated the limits, including frequent false-positive
outcomes, as a consequence of this enzymatic pre-
treatment (Hetrick et al., 2011; Park et al., 2012; Hart et
al., 2015; Szewczyk et al., 2016; Brown et al., 2017;
Alheim et al., 2018). It must be concluded, therefore, that
a standardized protocol has not been developed for this
procedure to date, suggesting that flow-cytometry-based
cr o s s-mat c hing ha s not yet ob t ained th e gener a l
938
Solid phase cross-matching prior to allo-grafting
acceptance necessary to substitute for, or ro utinely
supplement, the CDC-XM. For this reason, only a few
tissue-typing laboratories (especially among those of the
Eu r o trans p lant c o mmunit y ) actu a lly em p loy th i s
procedure.
Abo u t ten y ears a g o , an alternat i v e blo c k i n g
procedure was adapted to flow-cytometry-based cross-
matching which already had gained broad recognition by
avoiding artefacts and consequently led to improved
outcomes in immunohistochemical applications (Hajeer
et al., 2009). Hajeer and co-workers used heat-denatured
rabbit serum in order to reduce the background caused
by non-specific IgG-binding through its Fc-domain. In
this context it is noteworthy that several suppliers of
flow-cytometric diagnostic equipment have already for a
number of years offered commercial kits in order to
block Fc-receptors expressed on target cells or target
tissues using the same functional principle as in the
context of cross-matching first published by Hajeer and
co-workers. Generally, it is puzzling that this otherwise
well-established procedure was not introduced earlier
into this field of transplantation diagnostics in order to
re plac e t he use of pr onas e, whic h f or the reason s
discussed above seems to be rather a pseudo-scientific
approach.
In s pite o f this t e c h n ical a d v a n c e , a s t riking
dis a d v a n tage o f the FA C S-XM, w h i c h is also i n
comple te accord with the CDC-XM, is the general
dependence on a high cell quality, i.e. on a high degree
of vital cells, in both assays. As these requirements are
often not fulfilled by the donors’ samples, novel solid-
ph ase- base d c ross matc h s yste ms were dev elop ed,
which, in addition to other benefits, worked
independently of cell quality. Furthermore, we could
show that, based on their technical design, solid-phase-
based c rossmatch as s a y s extend th e fields of
applications, which are generally not feasible through
the use of both classical cellular crossmatch assays,
CDC-XM and FACS-XM.
Tec h n i c a l ben e f i t s lea d t o val i d and re l i a ble
diagnostic outcomes of solid-phase cross-matching
not attainable by CDC-based standard procedure
In the last 15 years, four different solid-phase-based
crossmatch assays have become commercially available,
three of which are based on the technical design of an
ELISA and one as a Luminex-based assay. They all have
been exhaustively tested in our laboratory between 2005
and 2019. As shown below, the first highly efficient
ELISA-based assay, which served as a valuable tool in
order to find adequate solutions for many patients, was
rep l a c ed by c o n s ecutive as say-base d systems of
increasing levels of inadequacy. This has led to the
current unsatisfactory situation that no solid-phase
crossmatch system of sufficient validity is available.
Thus, many of the diagnostic approaches discussed
below are generally no longer feasible and cannot be
carried out.
Solid-phase-based cross- matching as a solution for
allograft recipients pretreated with therapeutic antibodies
or cytostatic agents
Sol i d - phase-ba s e d cro s s - matching was first
described in the year 2005 in the context of CDC-based
crossmatch interferences through the use of therapeutic
humanized monoclonal antibodies (Book et al., 2005).
Book and co-workers investigated crossmatch outcomes
using CDC- and flow-cytometry- based techniques,
which were either completely or partially influenced by
the recipients’ sera after application of rituximab (anti-
CD2 0 ) , basilixi m a b / Simulect ( a n ti-CD25) a n d
al e m tuzuma b / Campat h (a n t i-CD52 ) . Th e au t h ors
con f i r med previ o u s observa t i o n s regard i n g the
artificially positive outcomes of CDC- as well as flow-
cytometric cross-matching after the administration of
alemtuzumab/Campath (Lyon et al., 2001; Wagenknecht
et al., 2004). Book and co-workers in their ground-
breaking study first systematically described an ELISA-
based crossmatch assay as a suitable tool to circumvent
the falsifying influence of applied therapeutic antibodies.
As the assay was termed Transplant Monitoring System-
(TMS-) ELISA (formerly GTI, Waukesha, USA) we
were unaware of this group’s investigations until 2013
when we serendipitously discovered their publication.
Thus, using the down-scaled second-generation system
ter m e d M i c ro A n tibody Monit o r i ng S ystem-
(MicroAMS-) ELISA (GTI, Waukesha, USA; FDA-No.
BK0 6 0 0 38 awar d e d on 26t h o f June, 2006; la t e r
Immucor, Stamford, USA) the idea to implement a solid-
pha s e - based crossm a t c h as s ay in th e co ntext of
rituximab-induced AB0-bloodgroup incompatible living
kidney donations arose independently in our laboratory
and was initiated in 2006 (Schlaf et al., 2012a,b, 2014a).
A scheme of this assay’s workflow is given in Fig. 1.
Briefly, a detergent-treated lysate of a given donor’s
tissue sample is pipetted into the wells of ELISA strips
pr e -coat ed w ith mAb, whi c h a re d irect e d a gains t
mo nomo rphi c e pito pes of HL A-cl ass I or clas s I I
molecules (Fig. 1A). After consecutive washing steps
the wells are incubated with the sera of the recipients
under investigation. These sera may contain the donor-
specific antibodies to be detected (red arrow) as they
serve as detection antibodies in this sandwich assay by
recognizing the immobilized donors’ HLA molecules
(Fig. 1B). After additional washing steps, the samples in
the wells are then incubated with secondary alkaline
phosphatase-conjugated anti-human IgG antibodies in
order to recogniz e the immo bilized donor-specific
antibodies (Fig. 1C). Early in the development of the
procedure we modified this final incubation step by
using secondary antibodies directed against IgG/M/A
isotypes of the primary DSA, as these isotypes are
known to be relevant in the context of allograft rejection
(Arnold et al., 2008, 2013). Furthermore, due to their
complement-activating feature DSA of the IgM isotype
are particularly readily detected using the CDC-based
crossmatch but not using standard commercial IgG-
939
Solid phase cross-matching prior to allo-grafting
specific antibody detection or identification assays. In
order to validate the data three controls are included: i)
the Lysate Control Reagents (LCR) consisting of a
se cond e nzym e-label led mA b f or the detect ion of
immobil ize d HLA class I or class II mol ecules b y
recognizing a second monomorphic epitope on the
bound HLA-molecules. It is important to note that a
sufficient amount of immobilized HLA-molecules is a
prerequisite to obtain a clear positive signal and this is
provided by this approach (Fig. 1D). ii) An additional
positive control in which the reagents used are tested
(not shown), consisted of adding freeze-dried control
lymphocytes and serum samples that are positive for
anti-HLA class I or class II antibodies, and these are
components of the kit. After rehydration of the cell pellet
an d it s a p plic a tion as ant i gen sour ce t his cont rol
demonstrates the functionality of the reagents provided
by the supplier, even if the preparation of the individual
donors’ tissues is inadequate. iii) A negative control,
which corresponds to the positive reagent control, with
the difference that an irrelevant serum negative for
HLA-antigens is used. The value of the recipient’s serum
under investigation had to exceed two-fold the value of
the negative control to be classified as positive. Taken
together, the network of controls in all cases under
investigation allowed categorization of the raw data as
valid or not and additionally pointed to the sources of
any errors.
Until 2012, all four kidney transplant centers for
which we put the prior-to-transplant crossmatches into
practice participated in AB0-bloodgroup incompatible
li ving kid ney dona tions . C onseq uent l y, a ll of t he
rec i p i ents were pr e -conditi o n e d with an t i - CD20
rituximab always leading to highly positive NIH-derived
scores of 6 to 8 for CDC-cross-matching with isolated
B-cells and scores between 2 and 4 with PBL, depending
on individually varying fractions of B-cells. Thus, in all
cases described the complement-activating potency of
rituximab, which belongs to the IgG1 isotype finally
leading to its B-cell-depleting activity, was always
monitored instead of donor-specific antibodies. Gatault
and co-workers confirmed these investigations by their
observation that rituximab, even at low concentrations
(i.e. inferior to 1µg/ml), has the potential to falsify B-
940
Solid phase cross-matching prior to allo-grafting
Fig. 1. Flow-chart of the AMS-ELISA for the detection of donor-specific HLA class I molecules (lysate procedure). A. Binding of the donor’s solubilized
HLA class I molecules by monoclonal capture antibodies that recognize a monomorphic epitope on HLA class I molecules. B. Binding of the donor-
specific anti-HLA antibodies (red arrows) to be detected from the recipient’s serum to the HLA molecules of the donor. C. Binding of alkaline
phosphatase-conjugated secondary antibodies to the recipient’s bound donor-specific anti-HLA class I antibodies and subsequent color reaction. The
original protocol was modified by substituting the human IgG-specific by a human IgG/M/A-specific secondary antibody. D. Lysate control using an
alkaline phosphatase-conjugated monoclonal antibody directed against a second monomorphic epitope as detection antibody in order to confirm the
immobilization of a sufficient amount of HLA molecules by the solid phase-bound capture antibody. The AMS-ELISA variant for the detection of donor-
specific antibodies directed against HLA class II molecules is designed in a similar manner.
cell cross-matching over the broad window of 30-120
days after treatment prior to transplantation. In two case
re p orts data were fir s t pr ovided on a d ose-e ffect
relat ion shi p lead ing to p ositive B -ce ll cro ssm atc h
outcomes up to about 80 days after the last application of
this antibody (Gatault et al., 2013).
Fur t h e rmore, an u n expected positive B-cell
crossmatch was reported as a consequence of rituximab
tre a t m ent in the conte x t o f s e vere idiopa t h i c
thrombocytopenic purpura of a given donor 12 days
prior to organ harvesting (Desoutter et al., 2016). Thus,
the authors p resented a cas e allowin g no ot h e r
conclusion than the treatment of a given donor with
rituximab antibodies also can lead to artificial CDC-
bas e d cr o ssmatch outc o mes; t hese data were not
supported by any other reference investigation, such as
the identification of DSA by virtual cross-matching.
This aspect of false-positive cross-matching was also
ob s erved for the ther a peutic ant i -CD25 anti b ody
basiliximab/Simulect. Basiliximab is directed against the
alpha-chain of the interleukin 2 receptor (CD25) and was
found to influence the results of CDC-based cross-
mat c h i ng more or less evenl y i n a l l t h r ee c ell
populations under investigation leading to an artificial
CDC- XM score of about 4 (Schlaf e t al., 2012a,b,
2014a; Schlaf and Altermann, 2017).
It i s notewo rth y that false-positi ve cross mat ch
outcomes, even though usually at higher concentrations,
also are observed for flow-cytometric cross-matching
when performed after the application of therapeutic
antibodies, as was shown for alemtuzumab (campath)
(anti-CD52), rituximab (anti-CD20) and daclizumab
(ba s i l iximab) (anti-C D 2 5 ) (B o o k et al., 2005;
Guillaume, 2018). In all cases, however, the alternative
ELISA-based c rossmat ch assay (TMS-ELISA) was
never affected by any therapeutic antibody, clearly
indicating the general advantage of using cell-free solid-
phase systems over any cellular, i.e. CDC- or FACS-
based, crossmatch procedure (Book et al., 2005).
Patients suffering from various forms of leukemia,
and who are, therefore, destined for allogeneic stem cell
tr ansp lant ation , g ener ally do no t h ave to ful fill a
negative crossmatch as a given recipient and her/his
ch o s en d o nor m ust b e H L A -ident i c al a t th e hi g h
resolution/four-digit level of resolution. However, in the
past few years we have increasingly been receiving
recipients who: i) had been treated by thrombocyte
donatio ns as a consequenc e of their anti-leuke mia
therapy; and ii) due to a lack of HLA-compatible stem
cell donors were destined for so-called haploidentical
stem cell donations e.g. arranged between parents and
their children or siblings exhibiting this decreased
degree of HLA-compatibility. Such configurations may
lead to the detection of DSA representing a contra-
indication for the intended stem cell transfer and the
requirement of a crossmatch to exclude them. In nearly
all of these situations, CDC-cross-matching did not
al l ow t he e xclusi on o f DS A, a s al l re sults (PB L ,
separated T- and B-cells) were doubtfully (score of 2) or
weakly (score of 4) positive. We concluded that the
positive reactions were due to an unspecific cell death
induced by the cytostatic agent 6-mercaptopurine used
for the anti-leukemia thera py. The f ind ing s of the
alternatively performed AMS-ELISA, however, were in
complete accord with the corresponding virtual cross-
matching and in no case exhibited DSA (Schlaf et al.,
2012a).
Solid-phase-based cross- matching as a solution for
allograft recipients suffering from underlying autoimmune
(immune-complex) diseases
In addition to the invalid outcomes of tests that can
occur in the presence of therapeutic antibodies or certain
drugs, the requirement t o substitute, or at least t o
complement, the CDC-based standard crossmatch has
increasingly been discussed over the last 10-12 years. In
particular, false-positive or -negative results using this
as say can be indu ced by the pres ence of imm u ne
com p l e xes in pr o s p e ctive re c i p i ents. Th e s e are
particularly readily detectable in patients who suffer
from underlying autoimmune diseases, mainly of the
immune-complex type (type III). Immune complexes
represent the first known sources of interference that
frequently lead to invalid CDC-crossmatch outcomes.
Notably, in an early study Ozturk and Terasaki reported
false-positive CDC-crossmatch outcomes as a result of
aut o - a ntibodie s and imm u n e co m p lexes such as
rheumatic factors (Ozturk and Terasaki, 1980). These
cytotoxic factors were at that time detected in patients
suffering from autoimmune diseases such as Systemic
Lupus Erythematosus (SLE) without any previous allo-
immu nization. Subsequently, Sumitran-Hol gerss on
described the frequent occurrence of artificially positive
out c o m es of C D C - based c r o s s-matchi n g as a
consequence of auto-antibodies and immune complexes
(cytotoxic factors) (Sumitran-Holgersson, 2001). The
approach to avoid these diagnostic artefacts through the
use of reducin g agen t s suc h as dithio t h r e itol/
dithioerythritol (DTT/DTE), although widely accepted in
the Eurotransplant community due to their selective
destruction of auto-antibodies of the IgM-isotype, has
been regarded for many years as ineffective. Sumitran-
Holgersson first described that autoantibodies generated
dur i ng a u toimmun e di s e ases such as S L E do not
necessarily belong to the IgM isotype but may also
belong to the complement-fixing (sub-) isotypes IgG1
and IgG3 (Sumitran-Holgersson, 2001). Additionally,
studies exist which refer to detrimental effects of HLA-
specific alloantibodies of the IgM-isotype, thus clearly
highlighting the diagnostic value not to destroy them
using reducing agents but to detect them (Vaidya and
Ruth, 1989; Stastny et al., 2009). Unfortunately, these
IgM alloantibodies, along with so-called weak (low titer)
IgG alloantibodies, can be eliminated by DTE/DTT,
despite still being readily detectable using solid-phase
cro s s m atch tech n i q u es modifi e d with second a r y
antibodies recognizing IgG, IgM and IgA isotypes of the
941
Solid phase cross-matching prior to allo-grafting
pri mary DSA . T hus, as men tione d a b ove we als o
introduced this modification into the workflow of the
AMS-ELISA as early as 2006 (Altermann et al., 2006;
Schlaf et al., 2012a). Based on the above arguments, the
general diagnostic approach of using reducing agents to
selectively eliminate autoantibodies and to specify HLA-
specific alloantibodies has been challenged for several
years. I t must be concluded that the appl ication o f
reducing agents, as with the use of pronase in order to
selectively digest Fc-receptors in the context of flow
cytometry cross-matching, must be regarded as a non-
scientific practice rather than an approach based on the
current state of scientific and technical knowledge.
To date, autoimmune diseases, and especially those
of the immune complex type (type III), represent the
most prominent disruptive factor for CDC-based cross-
matching leading to artificially positi ve outcomes.
Consequently, these artefacts lead to increasing numbers
of patients on the waiting lists for kidney allografts. On
the one hand, immune complex diseases are a frequent
reason for end-stage renal failure, but on the other hand,
th e un derly i ng disea s e-bas ed a rtifi ciall y po sitiv e
outcomes of CDC-based cross-matching result in this
test system being a major hindrance to the allocation of a
kidn ey allogra ft. In this context, we pub lishe d the
findings of several investigations that displayed ELISA-
based cross-matching as an adequate alternative in order
to validly indicate DSA and not disease-based interfering
factors (Schlaf et al., 2012a, 2013, 2014b, 2016). In spite
of this long-standing knowledge and our experience,
which we have gained over several years up to 2010, the
approach to circumvent artificial CDC-based crossmatch
results by considering the alternative valid solid-phase-
based crossmatch results was only followed twice in the
years 2009 and 2010 for legal reasons (Schlaf et al.,
2013, 2016). Strongly influenced by some
Eurotransplant authorities at that time, the German
Federal Medical Association in December 2010 defined
th e C DC-b ased cr ossm atch as th e only proc edur e
accredited for the allocation of post-mortem kidney
donations, whereas prior to this amendment the former
guidelines only claimed to “exclude the existence of
cytotoxic donor-specific anti-HLA antibodies” without
any methodical determination. Thus, the AMS-ELISA,
which was successfully used in order to exclude/detect
both cytotoxic and non-cytotoxic DSA (Schlaf et al.,
2013, 2016) and despite validly indicating the correct
gr afti ng in these two rep orte d c ases, is no long er
permitted. Consequently, we immediately stopped using
th is appr oach and wo rked in ac cord ance wi th the
updated guidelines, even though they are ineffective in
terms of their basis of the current state of knowledge in
immunology and may even be potentially harmful to the
patients. The increase in the number of positive CDC-
bas e d c rossmatc h a s says led t o t h e consequ e n t
accumulation of patients with the respective underlying
diseases from 6.5% in 2008 to currently at least double
this value (Altermann and Schlaf, 2010; Schlaf et al.,
2014c). The increasing number of patients who fail
CDC-b ased crossmatches and consequentl y do not
receive offered kidney allografts merely because of their
underlying autoimmune diseases, and not due to DSA,
should be the rationale to stop the erroneous methodical
determination imposed in 2010 and to accept additional
solid-phase-based cross-matching in this context. In
view of this drawback of CDC-based cross-matching,
coupled with the CDC-based antibody specifications
using cell tray analyses, both the decision of the German
Federal Medical Association and the corresponding
publication of a Eurotransplant group highlighting the
CDC as the leading procedure to define highly sensitized
patients must be regarded as wrong and completely
ineffective (Doxiadis et al., 2010).
Over the last nine years we have used solid-phase-
bas e d cro s s - matching , seve r a l ti m e s an d hig h l y
successfully, as a diagnostic approach for living kidney
donations in addition to the CDC-based crossmatch
out c o m es, w h i ch w e r e ma i n l y d u e to underly i n g
autoimmune diseases. Until now this has been possible
because the CDC-based crossmatch restrictions do not
ho l d t r ue f or a ccept ing or r efusi n g l i ving kid n ey
allografts. New guidelines are currently being prepared
by the German Federal Medical Association, which
hopefully will not lead to the prohibition of solid-phase
cross-matching in this additional context. Furthermore,
we have used it as a reference procedure in order to
identify false-positive CDC-outcomes, which may be the
result of temporary autoimmune attacks. These attacks in
some cases ease after their initial clinical appearance and
thus lead to some attack-accompanying sera, which
should be identified and rejected for upcoming CDC-
crossmatches. Thus, by defining the false-positive sera
to be rejected we were frequently able to accept kidney
offers for patients that would otherwise have been
refused due to exclusive artificially false CDC-based
out c omes (Schla f et al., 2014b ) . Th e pr o b lem o f
adequate kidney allocations, however, under the current
gui d e l ines rem a i n s irresol v a b le for pr o s pective
recipients who have consistently, or over a long period,
generated false-positive CDC-XM sensitive sera and for
patients on waiting lists who present for false-positive
cytotoxic factors in addition to anti-HLA antibodies.
Without doubt, these numbers on waiting lists will
continue to increase if solid-phase cross-matching does
not become available as a legitimate procedure (Schlaf et
al., 2016).
Solid-phase cross-matching using acellular donor tissue
such as corneal material, arterial vessel allografts or
stored donors’ cell lysates lacking single vital cells
Due to the fact that for corneal allografting neither
single nor vital cells are available for preparing detergent
lysates of donor tissue, we used the outer scleral rim of
cornea donors, which is generally available as a retained
sample after the excision of the inner part used for
allografting. Although this tissue is very poor in cells,
and these are se l f - evidentl y not is o latable as a
942
Solid phase cross-matching prior to allo-grafting
suspension of vital cells, the outer scleral rims actually
serve as adequate material for the Micro-AMS-ELISA in
order to explain preceding or to predict forthcoming
corneal rejections due to detected DSA (Altermann et al.,
2006; Sel et al., 2012). The same argument proved valid
for arterial vessel allografts as their tissue fragments
used as donor material are characterized by very similar
features. Thus, the Micro AMS-ELISA was also used by
us in order to detect DSA directed against HLA-antigens
of fresh or frozen arterial allografts (unpublished data).
Furthermore, we provided the first evidence that
sol i d - phase-ba s e d cross- m a t ching is suitab l e to
demonstrate an upcoming humoral immune reaction as
indicated by DSA. Deep-frozen leukocyte pellets from
blood or, due to its increased portion of HLA class II-
expressing B cells superior to blood-derived leukocytes,
es p e cially sple e n-deri v e d l e u kocyte pel l e ts w e re
successfully used as donor materials (Schlaf et al.,
2015a,b). This approach was followed using leukocyte
pellets, which had been deep-frozen up to 4.5 years and
most probably provides the feasibility to use cellular
material of post mortem organ donors stored for more
th an 10 year s. Thus, this EL ISA- base d p rocedur e
provides the option to routinely perform de facto cross-
matching using deep-frozen samples from deceased
donors in addition to virtual cross-matching; in other
words, the comparison of the recipients’ anti-HLA
antibody specifi citie s with the donors’ historically
identified HLA antigens.
To exclude, or to define, highly harmful DSA not
on ly v irtu ally but ad d itio nally by de fa c to c ross-
ma tching may be of speci al rele vance in case s o f
insufficiently documented historical typing results of
deceased donors, e.g. when the donor’s so-called split
antigens have not been adequately resolved or when so-
called allele-specific antibodies are present, as have been
in crea sing ly desc ribe d t o h ave arisen in an organ
recipient (Proust et al., 2009; Arnold et al., 2010; Schlaf
et al. , 2 0 12b; Pan d ey and Harvi lle, 2019 ). T hese
antibodies are not directed against the complete antigen
(e.g. HLA-A25) which genetically comprises a group of
alleles (e.g. HLA-A*25). Allele-specific antibodies
belonging to the same group of alleles, which a given
donor shares with her or his recipient (i.e. who both have
be e n t y ped HLA-A * 25) virtu ally appe a r a s au t o-
antibodies (e.g. r ecipient’s anti-HLA- A25 directed
against donor’s HLA-A25) if exclusively defined at the
two-digit (one field) level, which currently represents the
gui d e l ine-comp l i a nt r e q u irement for solid o rgan
transplantations. Thus, allele-specific DSA are virtually
undefinable at this level of resolution. In spite of such a
given low resolution-matched combination a recipient
char acterize d by the rare allele HLA-A*251 4 may
develop antibodies against the common allele HLA-
A*2501 shared by the vast majority of the HLA-A25
be aring pa tien ts, as both al leles pr esen t di ffe rent
ep itop es (Sch laf et al. , 2 012b ). Thes e a ntib odie s,
however, were clearly demonstrable through the use of
the donor ’s leukocyte pellet for retrospective solid-
phase-based de facto cross-matching. It is noteworthy
that this positive crossmatch result was subsequently
confirmed by virtual cross-matching at the high (four
digi t / two field) level o f res olution of typi ng and
corresponding high resolution antibody specifications.
These were performed using the so-called single antigen
level compri sing the majo rity of well- document ed
re c ombi n antly ge nerat e d a nd i mmobi lized al l elic
antigens.
Of course, this specialized application requires that
donor material is available and thus it would be highly
advantageous to establish systematically something akin
to a deceased donors’ tissue bank. Historically, the
extremely useful approach of implementing a de facto
crossmatch using deceased donors’ materials has only
onc e bee n fol l o w ed in th e co n t e xt of co r n eal
transplantations of high risk patients and this was over
two decades ago. Th e oph t h a lmologis t s and
immunologists involved used retinal pigment epithelial
cells isolated from explanted eyes and afterwards stored
them in liquid nitrogen. After thawing the cells they had
to be re-cultured and stimulated with IFN-γ in order to
upregulate the surface expression of HLA molecules for
the subsequent flow-cytometric crossmatch analysis
(Baumgartner et al., 1992; Zavazava et al., 1996).
However, this historical approach requires the use of
vital cells and is characterized as time- consuming ,
ex p ensiv e an d e x treme l y c h allen ging in terms of
technical demands. In con trast t o the p rocedure of
Zavazava and co-workers, which is inappropriate as a
routine method in most tissue-typing laboratories, the
ELI S A - based te c h nique pr e s e nted he r e is eas i l y
implementable in any laboratory without complex or
expensive technical equipment.
Discontinuation of the AMS-crossmatch ELISA and its
sub s e q u e nt rep l a c e ment by d i agnosti c s y stems
characterized by increasing insufficiencies, including
complete diagnostic failure
Various fields of diagnostic application all have
clearly demonstrated the superiority of solid phase-based
cross-matching over the CDC-based procedure and it
was therefore a signficant setback when this highly
reliable solid-phase assay was suddenly discontinued by
the manufacturer for commercial reasons in 2013. After
the announcement, there was a limited time-period of
only four weeks to establish and optimize the alternative
AbCross ELISA (manufactured by MicroCoat, Bernried,
Ger m a n y a n d distr i b u t ed b y Biora d , Münch e n ,
Germany) in a technically modified manner, which
de viat ed s igni fica ntly fr om t he orig inal pr otoco l.
According to the original AbCross protocol (presented in
Fig. 2) the binding of the recipient’s DSA had to be
fulfilled using intact and detached lymphocytes of the
respective donors (Fig. 2A). Subsequently, the resulting
complexes of HLA-antigens and DSA were isolated by a
det e rgent-me d i a ted lys i s step fo l l owed by
immobilization of immune complexes to the pre-coated
943
Solid phase cross-matching prior to allo-grafting
monoclonal capture antibodies specific for either HLA
class I or class II molecules (Fig. 2B). Afterwards, in
com p l e te accordan c e wi t h the AMS-ELI S A , th e
immobilized immune complexes were recognized by
DSA if present in the recipient’s serum. These were
fin a l l y visualiz e d us i ng an enzyme- c o n jugated
secondary anti-human IgG antibody (Fig. 2C). Also in
accordance wi th the AMS-ELISA, a nd as po sit ive
control, a second monoclonal antibody directed against a
second monomorphic epitope provided evidence that a
sufficient quantity o f immune complexes had been
ext r a c ted fr o m the donor ’s tissue sample and
immobilized to generate a clear signal (Fig. 2D). This
control was termed “Lysate Control” using the scheme
of the Micro - A M S EL ISA. The resultin g ra t h e r
laborious procedure, i ncl uding the initial de nsi ty-
gra d i e nt centrif u g a t ion step in ord e r t o i solate
lymphocytes, took at least six hours. Furthermore, a
large volume (20 ml) of donor blood, which is seldom
available from any given live donor and is an even more
significant problem when dealing with deceased donors,
was required in order to isolate a sufficient number of
HLA-class II antigen-expressing cells (mainly B-cells).
Despite these drawbacks Süsal and co-workers used this
original protocol and highlighted the superiority of the
AbCross method, especially in comparison to CDC-
based B-cell cross-matching (Gombos et al., 2013).
However, the conclusions drawn by the authors that the
better predictive value for a 2-years post-transplant graft
lo ss is due to a hig her sensit ivit y o f t he AbCross
technique must be critically challenged because the
majority of the CDC-based crossmatch artefacts also
hold true for B-cell cross-matching (Schlaf et al., 2014b,
c). Thus, it is likely that the increased specificity of
HLA-class II in AbCross cross-matching is the reason
for this assay’s superiority.
Apart from circumventing CDC-crossmatch-specific
artefacts, which resulted from the artificial activation of
944
Solid phase cross-matching prior to allo-grafting
Fig. 2. Flow-chart of the original AbCross-ELISA for the detection of donor-specific HLA class I molecules (immune-complex procedure). A. Binding of
the donor-specific anti-HLA antibodies (red) to their target HLA-antigens on the surfaces of detached T-cells and B-cells, respectively, in a first step.
B. Immobilization of the detergent-solubilized HLA-class I immune-complex to the monoclonal capture antibody recognizing a monomorphic epitope on
HLA-class I molecules. C. Binding of the enzyme-conjugated secondary anti-human IgG (alternatively anti-IgG/M/A) antibody to the bound donor-
specific anti-HLA antibodies of the recipient and subsequent color reaction. D. Positive control consisting of an enzyme-conjugated second monoclonal
control antibody bound to a second monomorphic epitope (here anti-ß2 microglobulin in order to form immune complexes with all HLA-class I antigens,
which are then immobilized by the capture antibodies). The AbCross-ELISA variant for the detection of donor-specific antibodies directed against HLA
class II molecules is designed accordingly.
the complement system, the original AbCross protocol,
as well requiring detached cells in suspension, did not
provide any additional advanta ge over the cellular
crossmatch technique. Thus, all of the application fields
dealing with donor materials lacking single or intact
cells, but which were nevertheless successfully handled
using the AMS-ELISA (Altermann et al., 2006; Sel et
al., 2012; Schlaf et al., 2015a,b), were not practicable
us i ng t he o rigin a l A b Cross - proto c ol p rovid e d b y
MicroC oat/Bio rad. In particular, since using “non-
cell ular” samples from don ors repres ents a unique
feature of the application, the technical design of the
AbCross-ELISA had to be completely changed and
adapted before use to be in full compliance with the
workflow of the AMS-ELISA.
However, surprisingly, and again for commercial
reaso ns, the AbCross -EL ISA was disco nti nued b y
MicroCoat/Biorad at the end of 2016. This forced us
rapidly to find an alternative procedure. The possibility
of a relaunch of the AMS-ELISA was received early in
2017 and raised our hopes to resolve this problem. Based
on using the same set of diagnostic antibodies as the
Micro-AMS, the novel diagnostic system, now termed
Donor-Specific Antibodies/DSA (Immucor Transplant
Diagnostics, Stamford, USA), was manufactured again
as a microbead-based assay using the Luminex platform,
which is well known from various anti-HLA antibody-
screening and specification assays of two commercial
suppliers. Given the aim of establishing the DSA-assay
as the only remaining solid-phase-based crossmatch
system, it was systematically evaluated in our laboratory.
The resulting data, however, were rather disappointing
because the accordance between virtual crossmatch
results used as reference data and those of the DSA assay
were far too low (Bau et al., 2019). Of the overall
results, comprising 212 independent anti-HLA class I
and class II a n tibody specif i c a t ions and t h eir
corresponding DSA-assays, 69 of the virtually defined
cros smatch result s (32.5%) had to be classified as
divergent using the DSA-assay, whereas only 143 results
(67.5%) were classified to be in accordance with the
ass a y ’s outcom e . Based on t h e chos e n cohor t of
re c i pient s (n = 1 06) no l e ss t han 6 2 ( 5 8.4%) were
characterized by findings that were not supported by the
virtual cross-matching. It is noteworthy that this high
discrepancy was in all likelihood not due to the well-
known possible errors of virtual cross-matching, as all
selected donor-recipient combinations were
straightforward in terms of the specificities and signal
intensities of the assay (Bau et al., 2019). As the set of
antibodies involved was the same as that used in the
AMS-ELISA, the immunochemical “hardware” could
not have been the reason for the diverging results. The
problem arose primarily from insufficiencies in the
software, which had to classify the recipients’ raw data
as ei ther po sitiv e o r n egat ive for DSA . S o-ca lled
background-adjusted factors (BAF) were subtracted
from Mean Fluorescence Intensity (MFI) values
measured against the immobilized donor antigens. This
BAF generally represented a cut-off value that was
calculated using the values of the three control beads
Con 1 (coated wi th albu m i n ), Con2 (coated w i t h
glycoprotein IV) and Con3 (naked bead) for an equation
specific for each lot. The basis of the calculation was
unclear, i.e. not revealed in the manual supplied by the
comp any, but r esult ed in s o-cal led “adjusted MFI-
values”, which could not be validated. Apparently, a
recipient’s serum s amp le was always classified as
positive if two of the three adjusted MFI values were
pos i tive. Furthe r m ore, serum sample s lea d i ng t o
increased raw values of all three control beads were also
nearly always classified as positive. Generally, in terms
of immunochemical analyses, the integration into the
evaluation algorithm of the values for ‘naked’, untreated
Con3-beads must be critically challenged, because any
binding of antibodies to t he naked carrier material
should be prevented through a blocking s tep. This
con s i d eration ho l ds t rue, of course, fo r a n y
immunochemical assay. Apart from the fact that the
un d erlyi n g e v aluat ion s oftwa re i s a “bl a ck b ox”,
including values which are incomprehensible in terms of
immunochemistry, apparently no clinical evaluation has
ever been performed by the supplier. If this had been
done, then the unacceptable deficiencies, which have
been similarly commented on by colleagues from three
ot her ti ssue -ty ping labor ator ies , w ould have bee n
discovered prior to its commercialization.
At the moment only 20 remaining single AbCross
assays are still available in our laboratory, which will at
the latest be used up by the end of the first quarter of
2020. For this reason, we have tried to evaluate the new
ELI S A -based crossm a t c h s y s t em n a m ed X M a tch
(P rotra ns, Kets ch, Germ a ny), wh i ch first be came
available in 2019. Unfortunately, this most recent assay
has be e n designe d in strict ac c o r dance wit h th e
workflow of the original AbCross assay (Fig. 2) and
consequently shares all of its disadvantages, described
above in detail since again it does not allow the use of
donor material lacking detached or intact cells. Thus, the
approach has been re-initiated to adapt the system to the
workflow of the AMS-ELISA. Initial results, however,
do no t d emonstra t e a t e c h nically co nvincing
modification, as the sera of many recipients show strong
signals even without prior immobilization of donors’
HLA-antigens (unpublished data). Using the XMatch
system about 25% of recipient serum samples were
characterized by antibodies binding unspecifically to the
matrix or the blocking material thus leading to false-
positive results. A ddi tio nal experiments using t he
protein-G column-purified IgG fraction of those sera led
to the same outcomes, demonstrating that indeed IgG
antibodies lead to the observed artefacts. We must
therefore conclude that the XMatch system will likely
not be adaptable to the workflow of the AMS-ELISA
(Fig. 1), obviating its use for our laboratory’s routine
work. Our investigations for implementing the Xmatch
ELISA are currently ongoing.
Based on the unique features and various fields of
945
Solid phase cross-matching prior to allo-grafting
appl i c a tion o f so l i d -phase-b a s e d cr o s s -matchin g
successfully carried out in the past, we conclude that
remanufacturing of a reliable assay along the lines of the
hi s toric a l AM S-ELIS A is one of t he m ost u rge n t
challenges and requirements of current, evidence-based
transplantation immunology. In particular, this technical
challenge needs to be addressed immediately in order to
facilitate the speedy manufacturing of reliable diagnostic
solid-phase-based crossmatch systems.
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Accepted April 15, 2020
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