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CLINICAL DILEMMA

Fatal peri-operative hyperacute graft rejection duringheart transplantation related to infusion of red blood cellconcentrateAnne Kennel, PhD,a Sophie Mattei, MD,b Fabrice Vanhuyse, MD,b Barbara Proust, PhD,a

Xavier Tinard, MD,c Hugo N. Kaneku, MD,d Paul Terasaki, MD,d and Pascale Perrier, MDa

From the aHistocompatibility Laboratory and bDepartment of Surgery of Cardiovascular Diseases and Transplantation, NancyUniversity Hospital, Vandoeuvre-lès-Nancy, France; cEFS-Lorraine Champagne, Haemovigilance Service, Vandoeuvre-lès-Nancy,

France; and dTerasaki Foundation Laboratory, Los Angeles, California.

With the use of sensitive new technology, hyperacute graft rejection is currently reported as anextremely rare event due to prospective lymphocyte crossmatches and/or virtual crossmatches. In thiscase study we describe a case of fatal early graft failure after a cardiac transplant in a male recipientwho had no anti-HLA antibodies detected before transplantation, but who received, during hearttransplant surgery, a red blood cell concentrate containing high levels of graft-specific alloantibodies.In addition, we analyze the relationship between these alloantibodies and the occurrence of hyperacuteallograft rejection.J Heart Lung Transplant 2012;31:1230–3© 2012 International Society for Heart and Lung Transplantation. All rights reserved.

KEYWORDS:heart transplantation;hyperacute rejection;transfusion;HLA alloantibodies;TRALI

Hyperacute graft rejection is usually considered an im-munologic event occurring within minutes or hours of rep-erfusion of the graft, caused by the recipient’s pre-formedcirculating antibodies.1 These are either natural antibodiesto ABO blood antigens, or anti-HLA antibodies remainingafter previous blood transfusions, transplants or preg-nancy.2,3 Ensuring ABO blood group compatibility and per-forming prospective lymphocyte crossmatches and/or detec-tion of pre-existing specific HLA antibodies prior totransplant are the accepted methods for preventing hyper-acute rejection (HAR).4 Early allograft failure due to HARis now extremely rare.5

Herein we report the case of a candidate for heart trans-plant who developed severe HAR and fatal graft failureafter the passive transfer of specific donor anti-HLA anti-bodies in a single red blood cell (RBC) concentrate.

Reprint requests: Anne Kennel, PhD, Laboratoire d’Histocompatibilité–Bâtiment EFS, CHU de Nancy-Brabois, Rue du Morvan, 54500 Vandoeuvre-lès-Nancy, France. Telephone: 33038-315-48-65. Fax: 33038-315-48-67.

E-mail address: a.kennel@chu-nancy.fr

1053-2498/$ -see front matter © 2012 International Society for Heart and Lunghttp://dx.doi.org/10.1016/j.healun.2012.05.017

Case report

The patient was a 36-year-old male with type O rhesusD–positive blood and HLA typing of HLA-A*01, A*26;B*08, B*18; DRB1*03, DRB1*04; DQB1*02, DQB1*03.He underwent a cardiac transplant for ischemic heart dis-ease and had been listed for 6 months on the nationalregister of cardiac transplantation candidates. He had 4pre-transplantation evaluations for HLA Class I and II an-tibodies, and for antibodies to MHC Class I–related Chain A(MICA) using a highly sensitive technique (LABScreenmixed; One Lambda, Inc., Canoga Park, CA). None of thetested sera presented detectable antibodies. There was nohistory of blood transfusion prior to the transplantation.

The donor was a 39-year-old male with type O rhesusD–negative blood and HLA typing of HLA-A*01, A*68;B*15:23, B*37; DRB1*08, DRB1*13; DQB1*04, DQB1*06.

The patient was transplanted according to a biatrial tech-nique, as described by Shumway. During the transplantprocedure, he was transfused by two red cell concentratesand one fresh-frozen plasma to keep hemoglobin at an

acceptable level. The transfusions were done 10 minutes

Transplantation. All rights reserved.

1231Kennel et al. A Fatal Heart Transplant Rejection

before release of the cross-clamp. A few minutes later, theleft ventricle began to swell and was dusky on externalinspection. Progressively, the right ventricle developed thesame aspect. This aspect evoked a HAR (Figure 1). Despiteintensive resuscitation, the patient died on Day 2 post-operatively due to multiple-organ failure.

The diagnosis of HAR was made after pathologic exam-ination of the first endomyocardial biopsy samples taken atthe first gross signs of rejection and then confirmed aftercardiac transplantectomy. The first samples did not showinterstitial edema. Cardiomyocytes were not notably altered.Congestive vessels, filled with red blood cells, were ob-served without interstitial infiltration of mononuclear cells.Immunostaining revealed no significant C4d deposition.According to the 2004 grading system of the InternationalSociety for Heart and Lung Transplantation,6 the rejectionwould be classified as Grade 0. Pathologic analysis of thegraft revealed diffuse edema, hyperemia, numerous foci ofinterstitial hemorrhage, and extensive areas of necrosis as-sociated with extravasated neutrophils (Figure 2a and b).Immunostaining for C4d remained negative. The finalpathologic diagnosis was hyperacute vascular rejection,probably antibody-mediated.

To explain this HAR we conducted several retrospectiveanalyses. ABO typing was controlled for both the recipientand donor and no irregular agglutinin was observed in therecipient. As for the serum collected prior to surgery, the assayfor anti-HLA antibodies remained negative (LABScreenmixed). No specific antibodies against MICA or HNA (humanneutrophil antigen) were detected for the tested isotypes, IgGor IgM. The retrospective crossmatch was performed usingtwo different techniques (complement-dependent mi-crolymphocytotoxicity [CDC] and cytometric flux analysis[CMF]) and did not show reactivity between the serumsample and the donor’s T- and B-cells.

After unclamping of the aorta and before the first mac-roscopic evidence of HAR, the patient received three trans-fusion components, two red blood cell (RBC) concentratesand 1 unit of fresh-frozen plasma (FFP) from 3 different

Figure 1 Operative view of the heart transplant.

donors. Distribution timing is shown in Table 1. We postulated

that the cytotoxic antibodies were passively carried by atleast one of the infused components. Sera from Donors 1and 2 were positive for anti-HLA antibodies, whereas noantibodies were detected in the plasma of Donor 3. Anti-body specificities were identified in the blood donors (Table1). Low levels of HLA antibodies were found in the serumof Donor 1, but the two specificities identified were notdirected against the HLA antigens of the transplanted organ.However, Donor 2 showed a large panel of HLA Class Iantibody immunization at very high levels. Among thespecificities identified, two were specific anti-graft antigens(B37, B15).

The CMF crossmatch between the serum of Donor 2 andthe lymphocytes of the heart donor was, however, negative.Using the CDC assay for anti-HLA antibody detection, thecurrent serum of Donor 2 also showed no reactivity with themononuclear cells from a representative panel of 30 donors.Crossmatches between the serum of Donor 2 and the cellsfrom donors expressing the same cardiac graft antigens,namely HLA-B37 and HLA-B*15:23 allele, indicated nocytotoxic reactivity. To explain the discordant results be-tween the anti-HLA antibody specificities found in the se-rum from Donor 2 and the absence of reactivity of thisserum with various cells, we suggest that these antibodieswere non-specific for HLA antigens, but were directedagainst denatured antigens fixed on the LABScreen beads.

Figure 2 (A) Histologic changes showing extravasation of red

blood cells (B) and inflammatory infiltrates and tissue necrosis.

B *15:2

1232 The Journal of Heart and Lung Transplantation, Vol 31, No 11, November 2012

The beads were then processed by various proteases toselectively remove denatured antigens, according to a pro-tocol developed by the Terasaki Foundation Laboratory.HLA Class I antibodies were retested with these cleanedbeads in the serum of Donor 2 (Figure 3). After the “clean-ing” process, only high levels of anti-B37 antibodies per-sisted. We also identified anti-HLA antibodies on eluatesfrom the cardiac biopsy, using single-antigen ClassI–cleaned beads. Two graft-specific antibodies were ob-served (anti-B37 and anti-A68) in the eluates. The otherantibody specificities observed belonged to epitopes relatedto A68 or B37.

Discussion

To our knowledge, this is the first report of a fatal hearttransplant rejection after the transfusion of an RBC compo-nent that contained specific antibodies directed againstClass I HLA antigens of the graft. Macroscopic eventsevoke a HAR. The absence of C4d staining could be due tothe low sensitivity of the C4d marker used. Taken together,the repeated control of the absence of anti-HLA antibodiesin various sera samples of the recipient, the negative retro-

Table 1 Timing of Injection and HLA Class I Antibodies Prese

Donor Blood componentTiming of infusionbefore macroscopic rejection

1 RBC 25 min2 RBC 10 min

3 FFP 5 min

FFP, fresh-frozen plasma; RBC, red blood cells.aB*52:01, B *15:03 and B *15:13 may be related to the rare allele

Figure 3 Specific HLA Class I antibody intensity (normalizedvalue of MFI) in serum of Donor 2 using LABScreen singleantigen before (blue histogram) and after cleaning beads fromdenatured antigen (red histogram). Only B37 antibodies remainafter the cleaning process. NC, negative control; PC, positive

control.

spective crossmatch between the patient’s serum and thedonor’s lymphocytes, and the absence of a history of eventsinducing immunization in the recipient argue against thepresence of circulating anti-HLA antibodies prior to cardiacsurgery. Identification of anti-HLA antibodies specific tograft antigens in one of the injected blood components andthe timing between the infusion and the occurrence of mac-roscopic HAR suggest that the origin of eluate antibodieswas passive transfer via the transfusion of a single RBCconcentrate. The discordance of results between CDC as-says and the more sensitive single allele beads assay may bedue to the sensitivity levels of each method.

Among the immunologic risks presented by the transfu-sion is transfusion-related acute lung injury (TRALI),7 aclinical syndrome that occurs in a temporal relationshipwith the transfusion of blood products. The pathogenesis ofTRALI is complex and not yet completely understood, butone of the major putative etiologies is the presence of HLAantibodies in transfused blood components.8 Even a residualamount of antibody-containing plasma from the donor canbe responsible for TRALI. Thus, TRALI is most oftencaused by the FFP,9 but has also been described after RBCtransfusions.10–12 Win et al13 showed that residual plasmavolume, as little as 10 to 20 ml, may induce TRALI. In ourstudy, the imputative RBC concentrate contained about 10ml of residual plasma, with very high levels of HLA anti-bodies, specific to graft antigens. According to the mechan-ical assistance set up during surgery, the blood componentswere directly dispensed, via the pump, into the cardiac graftwithout prior dilution in the body. This suggests that a highconcentration of allogeneic antibodies were, at first, in con-tact with the graft tissue.

One measure to potentially reduce TRALI risk is eitherto screen HLA antibodies in blood donors or at least toexclude female blood donors with a history of pregnancy,due to the strong association between HLA antibodies andmultiparous women.14 In the case just described, the 2HLA-antibody-positive donors were men without a historyof alloimmunization. Such antibodies in non-alloexposedindividuals have already been reported15 and may representan immune response to environmental antigen exposureinducing HLA cross-reactive antibodies, termed “natural”

he 3 Blood Donors

HLA Class I antibodies

ber of specificities DSA specificity Normalized MFI value

No 1,013 and 1,375Anti-B*37:01 15,414Anti-B*52:01a 12,053Anti-B*15:03a 12,845Anti-B*15:13a 11,298

3 of the donor according to the serologic assignment.

nt in t

Num

218

0

antibodies.16,17 Among the 18 specificities found in Donor

1233Kennel et al. A Fatal Heart Transplant Rejection

2, only anti-B37 (directed against graft antigen) remainedafter protease processing of beads, suggesting that this spec-ificity was directed against exposed epitope on native anti-gens and that all the other specificities were directed againstdenatured antigens or cryptic antigens. No explanation wasfound for the presence of this antibody in the healthy non-transfused donor.

Although low levels of HLA antibodies in blood com-ponents can lead to TRALI, there are, to our knowledge, nopublished data questioning whether such antibodies couldpromote allograft dysfunction if transfused into transplantrecipients. Until further studies confirm our hypothesis, it ispremature to suggest any particular course of action regard-ing the disposition of HLA antibody–containing blood com-ponents.

Disclosure statementThe authors have no conflicts of interest to disclose.

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