Surveillance of alloantibodies after transplantation identifies the risk of chronic rejection.

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by  Kimball, Pamela M

http://www.kidney-international.org original article
&2011InternationalSocietyofNephrology
Surveillance of alloantibodies after transplantation
identifies the risk of chronic rejection
Pamela M. Kimball1, Melissa A. Baker1, Mary B. Wagner1 and Anne King1
1Department of Transplant Surgery, Virginia Commonwealth University Health Systems, Richmond, Virginia, USA
The monitoringofthe levels ofalloantibodies following Despiteimprovementsinshort-termrenalallograftoutcome,
transplantation might facilitate earlydiagnosis of chronic long-termgraftsurvivalisunchanged,withchronicrejection
rejection(CR), the leading cause ofrenal allograftfailure. (CR) remaining the leading cause of graft failure.1–4
Here,we used serial alloantibody surveillance to monitor Compelling evidence indicates that circulating alloantibody
patients withpreoperativepositive flowcytometric againstdonorHLAantigenscontributestotheprocessofCR.
crossmatch(FCXM).Sixty-nineof 308 renal transplant For this reason, patients elaborating alloantibody before
patientsinourcenterhadpreoperativepositiveFCXM.Blood transplant generally exhibit poorer graft survival than
wascollected quarterlyduring the first postoperative year nonsensitized individuals.5–13 Post-transplant detection of
and tested by FCXMand singleantigen bead luminometry, alloantibody is associated with a higher incidence of
more sensitive techniques than complement-dependent CR,4,14–23 and circulating alloantibody is found in most
cytotoxiccrossmatching.Distinct post-transplant profiles patients at the time of graft failure.4–5,16,19–20,23–26
emerged and wereassociatedwithdifferent clinical Itisbelievedthatpost-transplantmonitoringofalloantibody
outcomes. Two-thirds ofpatients showedcomplete might facilitate early diagnosis of CR. Unfortunately, a
eliminationofFCXMand solid-phaseassay reactions within paradigm for effective monitoring has not been established
1 year, had few adverse events,and a 95% 3-yeargraft and the burden of developing a protocol falls upon individual
survival. In contrast,the remaining third failed to eliminate laboratories.Itisnotclearwhichpatientsshouldbemonitored,
flow FCXM orsolid-phase reactions directed againstHLA when they should be monitored, what techniques are most
class I or II antibodies. The inferior graft survival (67%) informative, or what alloantibody parameters are markers for
with loss inthis latter groupwasprimarilydue toCR. CR. A systematic longitudinal assessment of post-transplant
Thus, systematicassessmentoflongitudinalchangesin changes in alloantibody among unselected patients is needed.
alloantibody levels, either by FCXM orsolid-phase assay, Beginning in 2005, we initiated a prospective study to
can help identify patients atgreaterrisk ofdeveloping CR. address some of these issues. Because patients with
KidneyInternational(2011)79,1131–1137;doi:10.1038/ki.2010.556; preoperatively positive flow cytometric crossmatch (FCXM)
publishedonline26January2011 have always had poorer graft survival at this center, we
KEYWORDS:chronicrejection;donor-specificantibody;flowcrossmatching; developed a surveillance template to follow them exclu-
post-transplantantibodymonitoring sively.6,27 Blood was collected quarterly during the first year
after transplant and alloantibody determined using FCXM
andsolid-phaseassay.Post-transplantantibodychangeswere
correlated with the 3-year clinical outcome.
This report summarizes our cumulative findings of long-
itudinal changes in alloantibody profiles among patients with
preoperatively positive FCXM. We observed two distinct post-
transplant profiles: complete versus no elimination of alloanti-
body. However, only the latter profile was associated with
development of CR and poorer graft survival. Identification of
acceptable and nonacceptable post-transplant alloantibody
profiles should help stratify patients in terms of the risk for
developing CR and promote early diagnosis.
RESULTS
Correspondence: Pamela M. Kimball, Department of Transplant Surgery,
Virginia Commonwealth University Health Systems, 737 North 5th Street, Patients
Suite100,Richmond,Virginia,23219,USA.E-mail:[email protected] Patients were first partitioned on the basis of whether
preoperative FCXM was negative or positive. Patient
Received 19 August 2010; revised 29 November 2010; accepted 7
December2010;publishedonline26January2011 demographics were similar (Table 1). Both groups were
KidneyInternational(2011)79,1131–1137 1131
original article PMKimballetal.:Post-transplantalloantibodysurveillance
Table1|Demographics ofpatients withpreoperatively reductions in FCXM, DSA, or non-DSA levels against class I
positiveor negative FCXM orIIduringthefirstpost-transplantyear.PreoperativeT-and
FCXMnegative FCXMpositive P-value B-FCXM levels were equivalent (P¼0.4). Despite transient
fluctuations in T-FCXM levels, the levels subsequently
n 239 69
rebounded. B-FCXM levels were unchanging throughout
Ethnicity the first year. Solid-phase testing showed a similar pattern
AfricanAmerican 70% 80% 0.1 of alloantibody persistence. Preoperative DSA against class I
Caucasian 30% 20% 0.1
or II were found in 60 and 40% of patients. DSA levels
Female/male 56/44% 51/49% 0.3 against both class I and II were unchanged throughout the
Firstgrafts 89% 89% 0.9
year.Preoperativenon-DSAagainstclassIorIIwerefoundin
Deceaseddonor 72% 80% 0.2
Age(years) 47±1 43±5 0.3 60 and 60% of patients and levels were significantly greater
ABDRMM 4.8±0.5 5.0±0.6 0.5 than DSA levels (Po0.001). Non-DSA levels against class I
T-PRA 22±29% 50±37% 0.01
andIIdeclined23and52%bymonth6.However,bymonth
B-PRA 14±30% 39±39% 0.03
12, reactivity against class I and II was either unchanged
Abbreviations: ABDR MM, HLA antigen ABDR mismatch, FCXM, flow cytometric
orincreased.Totalnon-DSAlevelsagainstclassIandIIwere
crossmatch;PRA,panel-reactiveantibody.
significantly greater among group II than group I by month
primarily African Americans receiving first grafts from 12 (Po0.001).
deceased donors. Age and gender distributions were equiva-
lent.BothwerepoorlyHLAmatchedwithdonors.TheTand Comparison ofpretransplant antibody characteristics
B panel-reactive antibodies were higher among the FCXM- Once groups were identified based upon post-transplant
positive group. In all, nine patients had positive FCXM alloantibody elimination profiles, we re-evaluated preopera-
because of non-HLA antibodies and were excluded from tive antibody characteristics hoping to identify features
subsequent analysis. All remaining patients with positive that would predict post-transplant antibody developments.
FCXMshoweddonor-specificantibody(DSA)andnon-DSA. Unfortunately, none were found. The group demographics
Post-transplant alloantibody profiles. Two post-transplant were equivalent. The donor characteristics were equivalent
antibody profiles were observed. The majority of patients (Table2).Panel-reactiveantibodydistributionwasequivalent
(group I, 65%) showed complete elimination of FCXM with most patients showing sensitization (Table 3). T- and
reactivity within the first postoperative year (Figure 1a–c). B-FCXM levels were equivalent between and within groups.
The T- and B-FCXM levels were equivalent preoperatively All patients had DSA and non-DSA. DSA frequency (1 to 2
and at all time points (P¼0.4). Both T- and B-FCXM per patient) was equivalent between groups. DSA distribu-
reactivity became negative in 71% patients by month 6 and tionwasequivalentbetween groupsandwasdirectedagainst
100% by month 12. Reductions in FCXM levels were classIamong60–70%ofpatientsorclassIIamong30–40%of
mirrored using single antigen bead identification of DSA patients.FewpatientshadDSAagainstbothclassIandIIand
and non-DSA. Pretransplant DSA was exclusively directed most patients had only one DSA. DSA levels against class I
against class I or II in 66 and 34% of patients. Pretransplant and IIwere equivalent between groups. Although subjective,
DSA levels were equivalent against class I and II (P¼0.06). weconsiderDSAmeanfluorescentintensityaslow(o1000),
DSA levels against class I and II declined 90% by month 6 moderate (1000–5000), or high (45000) strength. Thus,
andwereundetectablebymonth 12.Toestimate atotallevel patientshadmoderatetohighalloantibodystrengthsinboth
of non-DSA, all individual mean fluorescent intensities were groups. The frequency of non-DSA (7±3) directed against
combined into a single value for each patient. Pretransplant classIorIIwassimilarbetweengroups.Totalnon-DSAlevels
non-DSA against class I or II were found in 60 and 50% of against class I were equivalent between groups and greater
patients.Totalpretransplantlevelsofnon-DSAagainstclassI thanagainstclassII(Po0.001).Theonlyovertdifferencewas
and II were greater than DSA levels (Po0.001) and the elevated non-DSA levels against class II in group II
predominantly directed against class I (Po0.001). Total relative to group I (P¼0.001).
non-DSA mean fluorescent intensity levels against class I
and II were reduced 50–70% by month 6 and 80–90% by Clinical outcome
month 12. Patientandgraftsurvivalwerecomparedbetweengroupsand
Threepatientshadprofilesandclinicaloutcomesimilarto patients with preoperatively negative FCXM. The 3-year
groupIbutfailedtocompletelyeliminateDSAagainstclassI actuarial patient survival was equivalent (P¼0.5) between
by month 12. Although they are probably a subgroup of FCXM-negative patients (99%) and groups I (100%) and II
groupIinwhichantibodyeliminationproceedsmoreslowly, (100%). In contrast, the 3-year actuarial graft survival
we eliminated them from subsequent analysis because of differed (Figure 2). Graft survival was equivalent between
their small size and nonidentical profile. FCXM-negative patients (96%) and group I (95%, P¼0.5)
Post-transplant antibody profiles for the remaining but poorer among group II (67%, Po0.001).
patients (group II) differed markedly from group I (Figure Additional clinical outcome parameters were compared
1d–f). Group II failed to eliminate or show significant (Table 4). The frequency of acute cellular rejection was low
1132 KidneyInternational(2011)79,1131–1137
PMKimballetal.:Post-transplantalloantibodysurveillance original article
125 300
d. d.
s. 100 s.
hifts ± 75 hifts ± 200
el s 50 el s 100
n n
an 25 an
h h
C C
0 0
0 3 6 9 12 0 3 6 9 12
Months Months
5000 7000
4000
FI 3000 FI
M M
A A 3500
S 2000 S
D D
1000
0 0
0 6 12 0 6 12
Months Months
1,00,000 1,00,000
FI FI
M 75,000 M 75,000
A A
S S
D D
n- 50,000 n- 50,000
o o
n n
al 25,000 al 25,000
ot ot
T T
0 0
0 6 12 0 6 12
Months Months
Figure1|Longitudinalassessmentofchangesinflowcytometriccrossmatch(FCXM),donor-specificantibody(DSA),andnon-DSA
levelsduringthefirstpost-transplantyear.BloodcollectedquarterlyamongpatientswithpreoperativelypositiveFCXMwastestedby
FCXMandsingleantigenbeadluminometry.NinepatientswithFCXMreactivitybutlackinganti-HLAspecificityandthreepatientswith
atypicalgroupIprofilewereexcludedfromconsideration.GroupI(n¼33)showedcompleteeliminationofFCXM,DSA,andnon-DSA
within12months.GroupII(n¼15)maintainedFCXM,DSA,andnon-DSAlevelsagainstclassIandIIthroughoutthestudyinterval.FCXM
symbols:JT-FCXMchannelshifts;KB-FCXMchannelshifts.DSAandnon-DSAsymbols:’anti-classImeanfluorescentintensity(MFI);
manti-classIIMFI.GroupI:(a)FCXMchannelshifts±s.d.;(b)DSAMFI±s.d.;and(c)non-DSAMFI±s.d.GroupII:(d)FCXMchannel
shifts±s.d.(e);DSAMFI±s.d.;and(f)non-DSAMFI±s.d.
Table2|Patientand donorcharacteristics betweengroups I group II (43%, Po0.001) and responsible for 80% of graft
andII failures versus 0% in other groups. Although mean serum
GroupI GroupII P-value creatinine at 1 year was greater among group II (Po0.01),
there was considerable overlap with the ranges seen among
1. Daysondialysis 1497±1283 1738±1510 0.6
FCXM-negative patients and group I.
2. Typeofdonor
Living 15% 33% 0.1 Despite alloantibody persistence, only half of group II
Standardcriteria 78% 47% 0.03 experienced AMR and CR within the study interval. The
Expandedcriteria 2% 7% 0.4
remainder stayed AMR and CR free. A comparison of
Donationaftercardiacdeath 5% 13% 0.3
3. Coldischemiatime(min) 913.1±617.1 728.9±507.9 0.3 pretransplantalloantibodycharacteristicsshowednoobvious
differences in FCXM, DSA, or non-DSA levels between the
subgroups (Table 5). Acute cellular rejection occurred with
(p5%) among FCXM-negative patients and group I but equalfrequencybetweenthesubgroups(25vs20%,P¼0.1).
greateramonggroupII(21%,Po0.001).Antibody-mediated However, group II patients without AMRenjoyed the 3-year
rejection (AMR) was minimal (p3%) among FCXM- graft survival equivalent to group I (100 vs 97%, P¼0.9).
negative patients and group I but prevalent among group II
(43%, Po0.001). Rejection severity and timing to diagnosis DISCUSSION
of AMR was equivalent between all groups. Although AMR Thereisstrongevidencethatanti-HLAantibodiescontribute
occurredlaterthaninmanystudies,thedelaymayberelated tothedevelopmentofCR,theleadingcauseofrenalallograft
to our use of thymoglobulin induction in all patients as well failure.1,2,4–5,10,14–15,17–18,23–29 While it is hoped that post-
as maintaining high levels of tacrolimus and mycophenolate transplant alloantibody surveillance might facilitate early
mofetil. Diagnosis of CR was low among FCXM-negative diagnosis,therearefewguidelinesregardinghowmonitoring
patients and group I. In contrast, CR was prevalent among should be performed and results interpreted. This report
KidneyInternational(2011)79,1131–1137 1133
original article PMKimballetal.:Post-transplantalloantibodysurveillance
Table3|Pretransplantantibodycomparisonbetweengroups 1.00
GroupI GroupII
0.75
(1)n 33 15 val
vi
ur 0.50
(2)T-PRA s
o10% 26% 35% aft
11–80% 33% 41% Gr 0.25
480% 41% 34%
0.00
(3)B-PRA
0 10 20 30 40 50
o10% 67% 75%
Months
11–80% 26% 17%
480% 7% 8% Figure2|Actuarialgraftsurvival.Deathswithfunctioning
graftswerecensored.’Patientswithpreoperativelynegative
(4)FCXMchannelshifts±s.d. flowcytometriccrossmatch(FCXM;n¼239);mGroupI;.GroupII.
T-FCXM 104±9 127±88 Po0.001,graftsurvivalamonggroupIIversusgroupI.
B-FCXM 101±70 118±74
(5)NumberofpatientswithDSA
ClassI Table 4|Comparison of the3-year clinical outcomes
1DSA 61% 47%
FCXM-neg GroupI GroupII
41DSA 6% 13%
n 239 33 15
ClassII ACR 4% 5% 21%*
1DSA 34% 27% AMR 1% 3% 43%*
41DSA 0% 13% BanffI 66% 60% 55%
BanffII 33% 30% 33%
(6)DSAMFI±s.d. BanffIII 0% 10% 11%
ClassI 3830±1450 5773±1081 MonthstoAMR 17±13 14±5 13±3
ClassII 2607±1304 4800±536 CR 1% 0% 43%*
Graftfailure 3% 5% 33%*
(7)Numberofpatientswithnon-DSA
ClassI 48% 40% Failuredue
ClassII 45% 33% ToCR 0% 0% 27%*
Death 1% 0% 0%
(8)Totalnon-DSAMFI±s.d. 1-yearsCr(mg/dl) 1.4±0.7 1.5±0.5 2.9±3.1*
ClassI 75,248±23,342 85,917±44,500 Range 0.7–8.6 0.8–2.9 0.6–9.5
ClassII 14,609±9784a 38,116±15,591a,b
Abbreviations: ACR, acute cellular rejection; AMR, antibody-mediated rejection;
Abbreviations: DSA, donor-specific antibody; FCXM, flow cytometric crossmatch; CR,chronicrejection;FCXM,flowcytometriccrossmatch;sCr,serumcreatinine.
MFI,meanfluorescentintensity;PRA,panel-reactiveantibody. Patients were initially partitioned based upon preoperative FCXM reactivity.
Patients were divided into groups based upon post-transplant alloantibody The preoperatively positive FCXM patients were then regrouped based upon
elimination profiles. Nine patients with FCXM reactivity because of non-HLA post-transplantalloantibodyeliminationprofiles.PatientswithpositiveFCXMdueto
antibodies and 3 patients with atypical group I profile were excluded from non-HLAantibodiesandanatypicalsubgroupofgroupIwereexcluded.Clinical
consideration. FCXM and singleantigenbead testingon Luminex platform were outcomewasdeterminedfromchartreview.
performedasdescribedinthetext.MostpatientshadonlyoneclassIorIIDSA.In *Po0.001,groupIIvsgroupIandFCXM-negativepatients.
thefewpatientswithtwoDSAsagainsteitherclassIorII,theMFIisthestrength
index of the immunodominant specificity instead of the cumulative strength. In
contrast,totalnon-DSAantibodyburdenwascalculatedbycombiningallnon-DSA positive FCXM because of preformed anti-HLA antibody
MFIsintoasinglevalueforeachpatient.Unlessotherwiseindicated,databetween
andwithingroupswerestatisticallyequivalent(P40.5).PreoperativeDSAtesting who,atthiscenter,havepoorergraftsurvival.6,27Changesin
wasperformeduponpatientswithnegativeFCXM,butnoDSAswereidentifiedwith specificity techniques throughout the years made original
thetechnologiesusedatthattime.
aPo0.001,totalnon-DSAMFIgreateragainstclassIthanII. specificity data incomparable. To standardize the data, we
bP=0.001,totalnon-DSAMFIagainstclassIIgreateringroupIIthanI. reassayed patient serum for anti-HLA specificities using one
lot of single antigen beads on a Luminex platform and also
retested donor HLA for minor histocompatibility antigens
summarizes our experience in attempting to develop a not determined originally. Consistent with a recent report,
utilitarianandclinicallyinformativemonitoringparadigmby the enhanced sensitivity of single antigen bead testing
correlating clinical outcome with longitudinal changes in allowed identification of DSA and non-DSA in 100% of
FCXM and solid-phase assay reactivity occurring during the study patients.30
first post-transplant year. We intentionally excluded patients Temporal changes in alloantibody levels were consistent
with preoperativelynegativeFCXM who typicallyexperience whether measured using FCXM or solid-phase assay. Two
excellent graft survival and minimal CR.6–7,12,16 We also post-transplant profiles emerged and showed different
excluded nine patients with preoperatively positive FCXM clinical outcomes. Patients showing complete elimination of
because of non-HLA antibodies and a small subgroup of alloantibody enjoyed excellent graft survival and few
groupI(n¼3)withslowerantibodyelimination.Instead,we complications. In contrast, patients failing to eliminate
focused our attention upon patients with preoperatively alloantibody had poorer outcomes and higher incidence of
1134 KidneyInternational(2011)79,1131–1137
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Table5|Pretransplant antibody profiles between group II graft, we believe the first possibility is the more likely
patients who didordid notexperience AMR mechanism.
The secondpost-transplant profileidentifieda groupthat
WithoutAMR WithAMR P-value
failed to eliminate FCXM, DSA, or non-DSA reactivity
1. T-FCXM(channels) 94±49 141±48 0.5
against class I and II within the first postoperative year.
2. B-FCXM(channels) 193±29 117±25 0.07
3. T-PRA(%) 56±23 64±13 0.7 Persistenceofcirculatingalloantibodypredisposedthisgroup
4. B-PRA(%) 11±8 49±14 0.1 forahigherriskofAMRandCRandultimately poorergraft
5. DSAMFI±s.d.
survival. The critical question is why is the alloantibody not
ClassI 5601±3601 5950±1598 0.9
ClassII 4376±1376 4978±938 0.8 eliminated among group II as occurred among group I?
6. Totalnon-DSAMFI±s.d. Unfortunately, we do not have an answer. Group II did not
ClassI 86,070±84,080 63,720±27,800 0.7 demonstrate any unique antibody characteristics before
ClassII 15,120±12,870 39,250±15,040 0.4
transplant. However, group II demonstrated several unique
Abbreviations: AMR, antibody-mediated rejection; DSA, donor-specific antibody; post-transplant differences that either individually or collec-
FCXM, flow cytometric crossmatch; MFI, mean fluorescent intensity; PRA, panel-
reactiveantibody. tively may predispose this group for CR. First, only group II
Half the patients in group II experienced AMR and had a high graft failure rate failed to eliminate alloantibody, which suggests that activa-
mediated by chronic rejection. The other half did not experience AMR and
tion ofdownregulatory pathwaysareinhibitedinthisgroup.
demonstratedexcellentlong-termgraftsurvivalwithoutevidenceofCR.GroupII
was partitioned into patients who did or did not experience AMR and their Second,onlygroupIIwasunabletoeliminateDSAsdirected
preoperativeantibodycharacteristicswerecompared.Mostpatientshadonlyone
againstclassIandII.Numerousstudiesshowthatcirculating
classIorIIDSA.InthefewpatientswithtwoDSAsagainsteitherclassIorII,theMFI
isthestrengthindexoftheimmunodominantspecificityinsteadofthecumulative DSA against either class I or II are deleterious to graft
strength.Non-DSAMFIsareacumulativevaluederivedbycombiningtheMFIsfor survival. Some studies havesuggested that anti-classII DSAs
allofthenon-DSAsintoasinglecumulativevalue.
may more aggressively promote CR than anti-class I.10,15
Thus, the perseverance of anti-class II among group II may
CR. Following delineation of specific groups, pretransplant enhance the chances of developing CR. Third, only group II
antibody characteristics were compared with the hope that wasunabletoeliminatenon-DSAdirectedagainstclassIand
some distinguishing parameters would predict post-trans- II and maintained significantly high levels of non-DSA
plant outcome. However, similar to previous reports, against class I and II. The precise role of non-DSAs in
pretransplant characteristics proved inadequate indicators allograftsurvivalisunclearbutthereisgrowingevidencethat
of post-transplant developments.8,10,13 Before transplant, they may contribute to allograft destruction by crossreactive
groups were equivalent in terms of demographics, donor recognition of shared epitopes on the allograft. If this
characteristics,alloantibodylevels,andspecificities.Alloanti- occurred among group II, then the high cumulative burden
bodies against class I predominated in all groups. However, of non-DSA levels combined with sustained DSA levels may
groupIIdidshowagreatertotalamountofnon-DSAagainst escalate the probability of CR.
class II than group I. Interestingly, despite alloantibody persistence, nearly half
It has always been puzzling why only a fraction of of group II remained AMR and CR free and had excellent
presensitized patients actually develop CR.6–10,16,19–20 Our graftoutcome.Thisisconsistentwiththehistoricinabilityto
surveillance study provides a partial explanation. The show 100% causal relationship between detection of
majority of patients (65%) showed rapid reduction of both circulatingalloantibodyandactivedisease.Becausepretrans-
T- and B-FCXM levels within 6 months of transplant and plant alloantibody characteristics were equivalent between
complete elimination within 12 months. These results were these subgroups (as well as group I), they were useless in
mimicked using solid-phase testing that showed that DSAs forecasting post-transplant antibody profiles or clinical
and non-DSAs against class I and II were simultaneously outcomes.Ourdatasuggestthat(1)alloantibody persistence
eliminated. Elimination of circulating alloantibody trans- is not an automatic trigger for AMR and (2) AMR without
formed this group into a FCXM-negative population that antibody persistence is not an automatic trigger for CR
typically has excellent long-term graft survival and minimal (groupIpatientswithAMRhavenot yetdemonstratedCR).
CR.Thecauseofantibodyeliminationisunknown.However, Ourinterpretationisthatalloantibodypersistenceduringthe
one possible mechanism could be downregulation of anti- first year heightens the risk of AMR. However, alloantibody
body synthesis after transplantation. Considering the short aloneisaninsufficienttriggerforAMR.Theextendedperiod
half-life of immunoglobulin G, complete depletion of of alloantibody persistence before AMR detection is similar
circulating alloantibody within 6–12 months is entirely to the lag reported in other studies between de novo
possible if synthesis stopped soon after transplantation. alloantibody appearance and rejection. We suggest that
Simultaneous depletion of DSA and non-DSA suggests that perhaps certain minimum conditions must be met in order
the effect is global and not specific to DSAs. Alternatively, to initiate alloantibody-mediated tissue damage. Perhaps
alloantibody binding to the graft could artificially lower alloantibody must be composed of complement fixing
circulating levels below detection thresholds of assays. isotypesatsomeminimallevelinordertoshowlyticactivity.
However, as this would not explain the simultaneous Similarly,perhapsdonor HLAexpressionmust exist atsome
elimination of non-DSAs unless they also bound to the minimal density in order to be an adequate target.
KidneyInternational(2011)79,1131–1137 1135
original article PMKimballetal.:Post-transplantalloantibodysurveillance
Confounding variables such as ongoing immune activating PATIENTSAND METHODS
events (like cellular rejection or infection) or nonimmune- Patients
mediated allograft damage may boost inadequate alloanti- A total of 308 patients underwent renal transplantation. All
body/donor antigen presentation and trigger rejection. We had negative T and B cytotoxic crossmatches. FCXM was
suggest that if certain minimal conditions are not met, then performed before transplant. Excluding patients who had
AMR does not occur, and maintaining high levels of DSA undergone desensitization, 69 patients (22%) elaborated a
and non-DSA appears clinically irrelevant at least for the positive preoperative FCXM. For these patients, blood was
short term. On the other hand, AMR with persistent collected quarterlyduring the first post-transplant year. Serial
alloantibody must create a smoldering rejection that is specimens were unavailable for eight patients who were
difficult to completely eradicate and upon rebound leads to excluded from subsequent analysis. Clinical outcome for the
chronic disease. It is hoped that early detection would remaining 61 patients was obtained from chart review.
facilitate AMR reversal. As this study was a blinded protocol Immunosuppression consisted of thymoglobulin (1.5mg/kg
in which alloantibody tests were not reported, medical daily for 4 days), SoluMedrol (1g at the day of transplant,
intervention was based upon clinical dysfunction and thus 500mg on day 1, 60mg on day 2, tapered 5mg/day until
the earliest intervention was not possible. 20mg/day, then tapered 5mg each month to 10mg/day),
The heightened sensitivity of the FCXM relative to mycophenolate mofetil (1g b.i.d.), and tacrolimus (0.1mg/kg
cytotoxic crossmatching has always generated controversy daily). Graft failure was defined as return to dialysis. Deaths
concerningitsrelevanceandconcernsthatsomereactionsare with functioning graft were censored. Protocol biopsies were
falsely positive. In this study, false-positive FCXMs are notperformed.Biopsiesweredoneforclinicalindications(for
unlikely as we only included patients for analysis when the example, serum creatinine increased 425% above baseline,
preoperatively positive FCXM reactions were validated by nephrotic range proteinuria, delayed graft function). Rejec-
identifiable DSA. Similar to a recent report, we found that tions were biopsy proven with histological classification
use of single antigen bead testing by luminometry detected following Banff ’97 criteria with updates.28 AMR was based
DSA in virtually all patients with positive FCXM.30 uponhistologicalfindingsofperitubularcapillariesfilledwith
Our goal was to develop a post-transplant monitoring polymorphonuclear cells, C4d-positive stain in peritubular
protocolthatwasclinicallyinformativeandmanageablefrom capillaries (staining available starting 2007), histological
a laboratory perspective. We focused on the patient group evidenceofendothelialitiswithpositiveFCXM,and/orfinding
most problematic to this center; the presensitized patients ofDSAs.CRwasidentifiedbyinterstitialfibrosis,fibrointimal
with preoperatively positive FCXM. We identified two arterial hyperplasia of arterioles, tubular atrophy and glomer-
distinct post-transplant alloantibody profiles that exhibited ulosclerosis, or membranoproliferative glomerular changes.
different clinical outcomes. Identification of patients at AMR was treated with alternate day Cytogam and plasma-
greater risk of CR should facilitate earlier diagnosis. pheresis. The study was conducted as a blinded investigation.
The study results allow us to consider strategies to alter The results were not reported and did not influence clinical
the post-transplant course of patients with persistently management. All practices conformed to the institutional
positive FCXM and DSA. One strategy would be to avoid review boardguidelinesfor theprotectionofhumansubjects.
transplantingpatientswithpositiveFCXMandhighlevelsof
DSA. However, complete avoidance would deny transplants T-and B-FCXM
Multicolor FCXM was performed as previously described.27,29
tothemajorityofpatientswhowilldevelopagroupIprofile
Channels shifts X50 were considered positive for T-FCXM
that unfortunately cannot be predicted pretransplant. If
and B-FCXM.
transplantation proceeds, preemptive immunomodulation
(intravenous immunoglobulin, plasmapharesis, rituximab,
Velcade) at the time of transplant might promote antibody Solid-phase testing
depletion in patients who would develop a group II pattern. Pretransplant panel-reactive antibody was determined using
Perhaps drug tapering protocols should be reconsidered in FlowBeads(OneLambda,CanogaPark,CA).Becausevarious
patients with group II profile. We feel longitudinal monitor- techniques for specificity determination were used through-
ingismoreeffectivethantestingonlywhenthereisevidence out the years, datawere not comparable. Therefore, to make
of graft dysfunction. Implementing protocol biopsies with data comparable and more sensitive, we (1) retested donor
C4d staining would be informative. Antibody persistence or HLA using SSP (One Lambda) to identify minor histocom-
elevationcoupledwithrisingserumcreatinineorhistological patibility antigens (Cw, DP) not determined originally and
changes in the graft should be treated aggressively. Patients (2) retested serums collected pretransplant and 6 and 12
with persistent alloantibody who experience AMR would months post transplant using one lot of single antigen bead
likely benefit from antibody-depleting therapies (perhaps assay on Luminex platform. Specificities and mean fluor-
Velcade and rituximab) to reduce or eliminate alloantibody escent intensities were determined following the manufac-
burdenthat mightminimize AMRrecurrence.Last,alloanti- turer’s instructions (GenProbe, San Diego, CA). DSAs are
body monitoringshouldcontinuebeyond1yearforpatients specificities against donor HLA antigens. Non-DSAs are
with persistent antibody. specificities directed against HLA antigens other than the
1136 KidneyInternational(2011)79,1131–1137
PMKimballetal.:Post-transplantalloantibodysurveillance original article
donor’s. Positive FCXM in the absence of anti-HLA 15. CamposE,Tedesco-SilvaH,MachadoPetal.Post-transplant
anti-HLAclassIIantibodiesasriskfactorforlatekidneyallograftfailure.
specificities were considered non-HLA antibodies.
AmJTransplant2006;6:2316–2320.
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Alltheauthorsdeclarednocompetinginterests.
appearanceofHLAantibodiesandfailureofkidneygraftsinafiveyear
longitudinalstudy.AmJTransplant2007;7:864–871.
ACKNOWLEDGMENTS 20. ScornikJ,GuerraG,ScholdJetal.Valueofposttransplantantibodytests
Therearenosourcesofsupport. intheevaluationofpatientswithrenalgraftdysfunction.AmJTransplant
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