Successful Salvage Therapy by Second Haploidentical Allogeneic Hematopoietic Stem Cell Transplantation in a Severe Aplastic Anemia Patient Presenting Donor-Specific Anti-HLA Antibodies After Graft Failure From Matched Sibling Donor HSCT: A Case Report and Review

BackgroundHematopoietic stem cell transplantation (HSCT) is potentially curative for severe aplastic anemia (SAA). Graft failure (GF) remains a life-threatening complication after HSCT. Preexisting anti-HLA antibodies, especially HLA-specific antibodies (DSA), have been demonstrated as a risk of GF.Case presentationThis report describes a woman with acquired SAA who presented with anti-HLA antibodies and GF. After the treatment of anti-HLA antibodies, engraftment was achieved through a second alternative donor HSCT. This work complied with the Declaration of Helsinki and the Declaration of Istanbul.ConclusionsBased on our experience in treating this case, we hold that the presence of preoperative anti-HLA antibodies could discount the efficacy of HSCT and anti-HLA antibody screening should be performed before HSCT. Additionally, a second HSCT is feasible to prolong survival.     

Right and Left Inverted Lobar Lung Transplantation

Adult recipients frequently withdraw from living‐donor lobar lung transplantation because of the small size of donor grafts. The right lower lobe is 120% larger than the left lower lobe. We developed a novel surgical technique in which an inverted right lower lobe graft can be transplanted into the left thorax. The first patient was a 43‐year‐old woman with end‐stage idiopathic interstitial pneumonia. Her husband was the only eligible donor for living‐donor lobar lung transplantation. His right lower lobe was estimated to provide 45% of the recipient's predicted forced vital capacity, which would provide the borderline function required for living‐donor lobar lung transplantation. Since lung perfusion scintigraphy of the recipient showed a right‐to‐left ratio of 64:36, transplanting the right lower lobe graft into the left thorax and sparing the native right lung was considered the only treatment option. We simulated this procedure using three‐dimensional models produced by a three‐dimensional printer. In living‐donor lobar lung transplantation, all anastomoses were performed smoothly as planned preoperatively. Because of the initial success, this procedure was performed successfully in two additional patients. This procedure enables larger grafts to be transplanted, potentially solving critical size matching problems in living‐donor lobar lung transplantation.     

Donor-derived soluble HLA plasma levels can not be used to monitor graft rejection in heart transplant recipients

OBJECTIVE: Increased levels of both donor- and recipient-derived HLA class I molecules (sHLA-I) can be found in serum or plasma of transplanted patients during rejection. Earlier data indicate that levels of donor-derived sHLA-I (dsHLA-I) correlate better with graft rejection than total sHLA Class I (Zavazava N, Kraatz E, Gassel AM, Muller-Ruchholtz W. Plasma MHC class I expression in cardiac graft patients: donor-specific soluble antigen in a pre-sensitized graft patient. Transplant Proc 1991;23:2258-2260; Claas FHJ, Jankowska-Gan E, DeVito LD, et al. Monitoring of heart transplant rejection using a donor-specific soluble HLA class I ELISA. Hum Immunol 1993;37:121). Therefore, quantifying donor-derived soluble counterparts of HLA Class I (sHLA-I) in the plasma of the recipient may offer a new possibility for non-invasive monitoring of rejection after organ transplantation. METHODS: In an extended study with 34 heart transplant recipients, we used sHLA-I specific ELISAs to monitor donor-derived soluble sHLA-A2, -A3, -A9, -B7, -B12 and B51. RESULTS: The assays were sensitive enough to detect dsHLA Class I in plasma of the recipients. However, the levels of sHLA were not found to be a useful tool for monitoring rejection. Rejection was often associated with low levels of donor sHLA. The recent finding that antibodies can inhibit the detection of sHLA molecules might explain this discrepancy. In order to test this hypothesis, patient sera were screened for the presence of anti-HLA antibodies and the results were related to the donor-derived sHLA levels. Only in four out of 34 patients HLA Class I specific antibodies could explain the low sHLA levels during rejection. CONCLUSIONS: In heart transplantation increased donor-derived sHLA levels are not a suitable marker for rejection and that antibody formation can not explain these results. Therefore, monitoring rejection episodes on the basis of donor-derived soluble HLA molecules is not a realistic approach to decrease the number of biopsies after heart transplantation.     

A predictive test for the efficacy of intravenous immunoglobulin in the inhibition of alloantibodies

Preformed recipient HLA-specific antibodies can cause hyperacute rejection of a transplanted kidney if they are directed against mismatched donor HLA antigens. To avoid hyperacute rejection it is essential that recipient antibodies be identified during patient workup for transplantation and HLA antigens to which a patient is sensitized then be avoided when selecting a kidney donor for them. Absence of donor-specific reactivity is then confirmed by a pretransplantation crossmatch test.     

Hyperacute renal allograft rejection from anti-HLA class 1 antibody to B cells —antibody detection by two color FCXM was possible only after using pronase-digested donor lymphocytes

We present a report of a transplant recipient who lost her renal allograft from hyperacute rejection. This was secondary to a weak IgG anti-HLA class I antibody that was only reactive to donor B lymphocytes. This antibody was not detected in her pretransplant serum by the conventional complement-dependent cytotoxicity assays using donor blood lymphocytes. Pretransplant sera were analyzed retrospectively by two-color flow cytometric crossmatching (FCXM). It was difficult to determine if the recipient's serum contained an IgG antibody specific for HLA on donor B cells since IgG from control AB sera and pretransplant sera bound equally well to CD19 B cells. However, when donor lymphocytes were pretreated with pronase to digest the membrane receptor for Fc domain of IgG (FcγR) on non-T-cells, control IgG in AB serum did not bind to B cells and, hence, it was easy to detect binding of IgG (in pretransplant sera) to HLA on B cells. This case underscores the importance of identifying weak anti-HLA class I antibodies reactive only to B cells. Moreover, it shows that the currently used two-color FCXM lacks the specificity to detect such antibodies.     

Transfer of HLA‐Specific Allosensitization From a Highly Sensitized Deceased Organ Donor to the Recipients of Each Kidney

We report for the first time the adoptive transfer of donor HLA‐specific allosensitization in two recipients following kidney transplantation from a highly sensitized donor. Kidneys from a donation after circulatory death donor were transplanted into two nontransfused, HLA‐specific antibody negative males receiving their first transplant. Antibody screening 7 days after transplant showed high level de novo IgG HLA class I‐ and class II‐specific antibodies in both recipients, with largely overlapping antibody profiles but no antibodies to donor HLA. The unusually rapid appearance of de novo alloantibodies in immunosuppressed nonsensitized recipients and absence of donor HLA‐specific antibody prompted testing of stored donor serum that revealed high antibody levels with specificities very similar to those seen in both recipients, but in addition the presence of strong antibodies to each recipient HLA. Alloantibody levels gradually declined but were still detectable at 3 months. These findings suggest that alloreactive passenger B cells/plasma cells within the kidneys of highly sensitized donors may give rise to rapid development of posttransplant de novo HLA‐specific alloantibodies. While the clinical significance of this phenomenon is uncertain it provides one explanation for the appearance of de novo HLA‐specific antibodies directed against third party but not donor HLA.     

Anti-HLA antibodies in kidney transplanted patients

Anti-human leukocyte antibodies (HLA) play a central role in graft survival, particularly in kidney transplantation. The presence of preformed donor specific anti-HLA antibodies is always excluded before transplantation by performing crossmatches using current and historic recipient serum samples. Several recent studies have observed a correlation between HLA antibodies and graft rejection. It has been suggested that these antibodies should be monitored routinely after kidney transplant to predict graft failure. Here in report the results of a study of on serum samples from 111 kidney transplant recipients that were monitored for anti-HLA antibodies using flow cytometry. Anti-HLA antibodies were only detected in four pre-immunized patients and showed the same HLA specificity that was present before the transplantation (in two cases against previous graft antigens). Furthermore, only two patients with functioning grafts developed anti-HLA antibodies, at 1 month and 1 year after the transplantation. However, they were not donor specific, but probably related to posttransplant transfusions. In our study, none of the patients who suffered an adverse event during the first year (including two with histologically documented acute rejection) developed anti-HLA antibodies. These results are probably related to the use of mycophenolate mofetil, which may reduce the incidence of HLA antibodies. We cannot exclude the possibility that antibodies produced by some patients may not be detectable because they are attached to the graft.     

Panel reactive antibody positivity and associated HLA antibodies in Turkish renal transplant candidates

Pre- and post-renal transplantation panel reactive antibody (PRA) screening is associated with increased incidence of hyperacute or acute graft rejection and graft loss. This study was designed to find any relationship PRA sensitization and associated human leukocyte antigen (HLA)-specific antibodies in Turkish renal transplant candidates. We included 340 patients who were in the renal transplantation waiting list in the study. We determined PRA sensitization ratio and the associated anti-HLA IgG antibody distribution of the patient group. The PRA testing was currently performed and levels above 30% were accepted to be positive. The PRA class I positivity was determined in 24 (7%) and class II in 34 (10%) of the patients. The most frequent HLA antibodies for class I were B56, A2, A34, A1, A23, A24 and B61; and for class II were DR11, DR14, DQ7, DR10, DQ5, DR1 and DR7, respectively. From these, the increase of the numbers of anti-HLA class II antibodies was significantly correlated with the increase of PRA sensitization ratio. In conclusion, the identification of the associated HLA-specific antibodies and correlation with the Turkish population HLA antigen distribution will identify the high-risk patients who are candidates for transplantation.     

Perioperative assessment of oversized lobar graft downsizing in living‐donor lobar lung transplantation using three‐dimensional computed tomographic volumetry

A 15‐year‐old boy with bronchiolitis obliterans after bone marrow transplantation successfully underwent bilateral living‐donor lobar lung transplantation (LDLLT) with segmentectomy of the superior segment of an oversized right lower lobe graft. As the recipient was small for his age, the predicted value of his functional vital capacity of the recipient was difficult to determine preoperatively. Three‐dimensional computed tomography (CT) volumetry revealed that the ratio of donor graft volume to recipient hemithorax volume was 159% on the right side and 82% on the left side. The patient is alive and well 7 months after transplantation, and three‐dimensional CT volumetry revealed that the right and left donor lungs were still compressed to 73% and 84% of the original size, respectively. In LDLLT, segmentectomy of the superior segment of the lower lobe is a useful option for downsizing an oversized graft and three‐dimensional CT volumetry can provide meaningful data for size matching.     

Successful transatlantic bilateral hand transplant in a young female highly sensitized to HLA class II antigens

Vascularized composite allografts may be more susceptible to rejection than other types of organ transplants, particularly in sensitized recipients. We describe a successful transatlantic bilateral hand transplant in a 40-year old woman who was highly sensitized to class II HLA antigens including HLA-DPB1 (UNet CPRA = 86%). Prior to transplantation, we selected an upper limb donor based on HLA class II matching and absence of donor specific antibodies, given evidence that class II mismatches are associated with acute cellular rejection in hand transplants. The patient was conditioned using five doses of thymoglobulin, and her immunosuppression included tacrolimus, rapamycin, mycophenolate, and prednisone. Post-transplant, the patient non-DSA anti-HLA antibody levels drastically increased, but only transiently and weak DSAs developed, which became undetectable by two months posttransplant. Following transplantation, periodic biopsies over 6 months indicated no evidence of rejection except for transient Banff grade 1 and one sample with grade 2 acute rejection. There was no evidence of rejection on her recent 1-year follow-up. The patient is currently healthy, has recovered protective sensibility, and is regaining excellent function. This case highlights the importance of pre-transplantation planning, donor selection/compatibility, and ethical considerations in the ultimate success of VCA.     

Hyperacute renal allograft rejection from anti-HLA class 1 antibody to B cells -antibody detection by two color FCXM was possible only after using pronase-digested donor lymphocytes

Abstract We present a report of a transplant recipient who lost her renal allograft from hyperacute rejection. This was secondary to a weak IgG anti-HLA class I antibody that was only reactive to donor B lymphocytes. This antibody was not detected in her pretransplant serum by the conventional complement-dependent cytotoxicity assays using donor blood lymphocytes. Pretransplant sera were analyzed retrospectively by two-color flow cytometric crossmatching (FCXM). It was difficult to determine if the recipient's serum contained an IgG antibody specific for HLA on donor B cells since IgG from control AB sera and pretransplant sera bound equally well to CD19 B cells. However, when donor lymphocytes were pretreated with pronase to digest the membrane receptor for Fc domain of IgG (FcyR) on non-T-cells, control IgG in AB serum did not bind to B cells and, hence, it was easy to detect binding of IgG (in pretransplant sera) to HLA on B cells. This case underscores the importance of identifying weak anti-HLA class I antibodies reactive only to B cells. Moreover, it shows that the currently used two-color FCXM lacks the specificity to detect such antibodies.     

Selective omission of the donor cross-match before renal transplantation: efficacy, safety and effects on cold storage time

BACKGROUND: A donor lymphocyte cross-match (XM) test performed before renal transplantation is considered mandatory but may delay the transplant and increase the cold storage time. With careful documentation of sensitizing events and with knowledge of previous antibody screening results, it is often possible to predict the XM result for a given donor HLA mismatch. In this study, a policy was adopted of omitting the pretransplant XM in patients in whom a negative result was predicted with absolute confidence. METHODS: Recipients were selected for cadaveric donor kidney transplantation using a computer algorithm based on HLA match, sensitization status, time on the waiting list and donor and recipient age. The immediate pretransplant cross-match test was omitted in non-sensitized recipients and in sensitized recipients where antibody specificities were precisely defined and not against donor HLA. RESULTS: From October 1997 to May 1999, 53 of 96 (55%) consecutive cadaveric kidney donor transplants were performed without a pretransplant XM. In all cases, a negative donor HLA-specific antibody XM was confirmed after transplantation. Omission of the pre-transplant XM was associated with a significant reduction in cold ischemic time (15.0 hr vs. 18.2 hr, P=0.01) and a reduced incidence of delayed graft function (13% vs. 33%, P=0.03). However, there was no difference in transplant outcome at 1 year. CONCLUSION: Rigorous attention to priming events together with careful antibody screening allows the pre-transplant XM test to be safely omitted in approximately half the patients awaiting renal transplantation. This policy allows a modest reduction in cold ischemia time, but it remains to be seen whether this is of clinical benefit.     

Transfer of donor anti‐HLA antibody expression to multiple transplant recipients: A potential variant of the passenger lymphocyte syndrome?

Antibody‐mediated rejection, whereby transplant recipient B cells and/or plasma cells produce alloreactive anti‐human leukocyte antigen (HLA) antibodies, negatively influences transplant outcomes and is a major contributor to graft loss. An early humoral immune response is suggested by the production of anti‐HLA donor‐specific antibodies (DSA) that can be measured using solid phase assays. We report the early posttransplant coexistence of a shared anti‐HLA antibody profile in 5 solid organ transplant recipients who received organs from the same donor. Retrospective analysis of the donor's serum confirmed the presence of the same anti‐HLA profile, suggesting the transfer of donor‐derived anti‐HLA antibodies, or the cells that produce them, to multiple solid organ transplant recipients. The time frame and extent of transfer suggest a novel variant of the passenger lymphocyte syndrome. These findings have important implications for the consideration of all posttransplant antibody measurements, particularly the interpretation of non‐DSAs in the sera of transplant recipients.     

肾移植术后监测受者抗HLA抗体的临床意义

目的 监测肾移植受者术后抗HLA抗体水平,探讨新生的抗HLA抗体对移植肾功能的影响.方法 共有384例肾移植受者术后进行了抗HLA抗体的监测,监测时间3～96个月,所有受者术前的抗HLA抗体水平均为阴性.使用莱姆德抗原板,采用酶联免疫吸附法(ELISA)检测抗HLA抗体,抗HLA抗体水平＞10%为阳性.对术后抗HLA抗体阳性者与阴性者间移植肾功能进行比较,观察新生抗HLA抗体对移植肾功能的影响.结果 术后抗HLA抗体阴性者318例(82.8%);阳性者66例(17.2%),其中抗HLA Ⅰ类抗体阳性者3例,抗HLAⅡ类抗体阳性者61例,抗HLA Ⅰ类和Ⅱ类抗体均为阳性者2例.HLA-DR位点0个抗原错配者92例受者中有7例新生抗HLA抗体,1～2个抗原错配者292例中有59例新生抗HLA抗体,二者比较,差异有统计学意义(P＜0.01).术后抗HLA抗体阴性者中移植肾功能良好者占87.4%(278/318),抗HLA抗体阳性者中移植肾功能良好者占65.2%(43/66),二者比较,差异有统计学意义(P＜0.05).结论 HLADR位点的抗原错配与肾移植后抗HLA抗体的产生密切相关,而新生抗HLA抗体会造成移植肾功能下降,从而降低移植存活率,肾移植术后监测抗HLA抗体有一定的临床意义.

Abstract：

Objective To detect de novo development of anti-HLA antibodies after renal transplantation, and to investigate their influence on graft function. Methods 384 kidney recipients,who were negative for anti-HLA antibody before transplantation, were monitored for anti-HLA antibodies over a period of 3-96 months, and a sensitive enzyme-linked immunosorbent assay (ELISA) was used to detect anti-HLA antibodies. HLA antibody ＞10 % was defined as positive levels. Results Among 384 recipients tested, 318 recipients (82. 8 %) were negative for anti-HLA antibody after transplantation; 66 recipients (17. 2 %) developed de novo HLA antibodies, 3 recipients with HLA class Ⅰ, 61 with HLA class Ⅱ, 2 with both HLA class Ⅰ and Ⅱ. According to amino acid residue matching, 7 cases developed de novo antibodies among 92 recipients with 0 HLA-DR mismatches,compared with 59 cases among 292 recipients with 1-2 mismatches, which showed significant difference between two groups (P＜0. 01 ). 87. 4 % (278/318) recipients negative for HLA antibodies after transplantation achieved good graft function, in comparison with 65. 2 % (43/66) recipients positive for HLA antibodies (P＜0. 05). Conclusion De novo production of HLA antibodies posttransplantation may be closely associated with HLA-DR mismatch. De novo HLA antibodies posttransplantation might damage graft function and reduce graft survival rate. The detection of de novo development of anti-HLA antibodies after renal transplantation has clinical significance for assessing renal allograft function.     

动态分析HLA和MICA特异性抗体对移植肾功能的影响

目的 探讨肾移植前后人类白细胞抗原(HLA)和主要组织相容性一类相关链A基因(MICA)抗体特异性对移植后排斥反应和移植肾功能的影响. 方法采用免疫荧光液相芯片技术检测27例肾移植(尸供22例,亲属活体供肾5例)受者手术前后抗HLA抗体和MICA抗体的特异性和阳性值变化,并结合供受者的基因分型,区分供体特异性抗体和非供体特异性抗体.取同期的临床资料和SCr水平进行分析. 结果 27例移植患者中带肾存活26例,移植肾失功1例.移植后1、3、6、12个月时动态随访24例,失访2例.27例患者移植前预存抗体7例(25.9%),其中HLA抗体阳性2例、MICA抗体阳性3例,HLA和MICA抗体均阳性2例.肾移植前HLA和MICA抗体均阴性者中移植后3～6个月产生新生抗体3例.1例新生HLA-Ⅱ类特异性抗体者,移植半年后出现慢性排斥反应,经治疗术后1年SCr>200 btmol/L.3例肾移植前MICA抗体阳性者,术后MICA抗体的特异性均无改变,但抗体的阳性分值呈现2～8分的变化,1年后均升高到移植前(4～8分)水平.移植前预存低阳性率HLA-Ⅱ类特异性抗体者1例,移植后2周有发热等排斥反应,巨细胞病毒检测阳性,移植后1个月时SCr为171μmol/L,3个月升高到236μmol/L.24例分为抗体阴性组(14例)和抗体阳性组(10例).移植后1个月和1年时SCr水平2组间比较差异有统计学意义(P=0.03,0.05). 结论 移植后3～6个月是新生抗体变化的重要随访时间,可根据HLA和MICA抗体的特异性和阳性分值变化,尽早采取有效方法预防排斥反应和减少移植肾功能减退的发生和发展.     

Anti-HLA antibody binding to hla class I molecules induces proliferation of airway epithelial cells: a potential mechanism for bronchiolitis obliterans syndrome

OBJECTIVE: Development of anti-HLA antibodies is associated with development of bronchiolitis obliterans syndrome after lung transplantation. We sought to determine the mechanism by which anti-HLA antibodies affect the development of bronchiolitis obliterans syndrome. We postulated that anti-HLA antibodies bind to the donor lung epithelium and stimulate phosphorylation and proliferation. METHODS: The A549 lung epithelial carcinoma cell line was cultured in serum-deficient medium to produce static growth. Then the cells were treated with anti-HLA sera from lung transplant recipients, pooled anti-HLA serum from highly sensitized patients, or normal human serum. The cells were also treated with the W6/32 mouse anti-HLA class I monoclonal antibody or control mouse IgG. Tritiated thymidine uptake was determined at 24, 48, and 72 hours. In parallel experiments the cells were treated as described above, and the levels of tyrosine phosphorylation were determined by Western blot analysis. RESULTS: Cells treated with anti-HLA serum or the W6/32 monoclonal antibody exhibited significantly greater proliferation and tyrosine phosphorylation of proteins of approximately 170, 130, 110, and 70 kd compared with cells treated with normal human serum or mouse IgG, respectively. CONCLUSIONS: These data indicate that anti-HLA antibodies have the ability to stimulate airway epithelial cell proliferation and that they may play an important role in the development of bronchiolitis obliterans syndrome. Prevention of HLA sensitization and immunosuppression with agents capable of blocking indirect antigen presentation and the humoral immune response against the allograft may be pivotal in preventing the development of bronchiolitis obliterans syndrome after lung transplantation.     

Rejection of a kidney transplant does not always lead to priming of cytotoxic T cells against mismatched donor HLA class I antigens

BACKGROUND: Previous studies showed that graft rejection is often associated with the presence of primed cytotoxic T cells (CTLs) with a high avidity for donor cells. Similar high avidity CTLs have been found in individuals who have formed IgG anti-HLA antibodies. The presence of such CTLs to a specific HLA mismatch is therefore considered to be a reflection of an activated immune system, and a contraindication for retransplantation with a donor sharing this particular HLA class I mismatch. METHODS: In our study we investigated whether patients have always primed CTLs against all individual HLA class I mismatches present on a rejected graft. Therefore, 14 patients who had undergone transplantectomy after irreversible kidney graft rejection were analyzed with respect to donor-specific CTLp frequencies and the presence or absence of high avidity CTLs directed against HLA class I mismatches present on the rejected graft. RESULTS: Patients, who have not formed anti-HLA antibodies against the donor have mainly naive CTLs. Most of the patients, that have developed IgG anti-HLA antibodies against a donor mismatch, have primed CTLs directed against that particular mismatch. However, patients with IgM anti-HLA antibodies only, and patients with IgG anti-HLA antibodies in historical sera but no IgG anti-HLA antibodies in current sera, have mainly naive CTLs against the donor HLA mismatch. CONCLUSION: Our results suggest that it is not always necessary to exclude repeated HLA class I mismatches for a subsequent transplantation. In addition to good anti-HLA antibody screening, the CTLp-assay may be a useful tool for donor-selection in retransplant candidates.     

Predicted Indirectly Recognizable HLA Epitopes Presented by HLA‐DRB1 Are Related to HLA Antibody Formation During Pregnancy

Pregnancy can prime maternal immune responses against inherited paternal HLA of the fetus, leading to the production of child‐specific HLA antibodies. We previously demonstrated that donor‐specific HLA antibody formation after kidney transplantation is associated with donor‐derived HLA epitopes presented by recipient HLA class II (predicted indirectly recognizable HLA epitopes presented by HLA class II [PIRCHE‐II]). In the present study, we evaluated the role of PIRCHE‐II in child‐specific HLA antibody formation during pregnancy. A total of 229 mother–child pairs were HLA typed. For all mismatched HLA class I molecules of the child, we subsequently predicted the number of HLA epitopes that could be presented by maternal HLA class II molecules. Child‐specific antigens were classified as either immunogenic or nonimmunogenic HLA based on the presence of specific antibodies and correlated to PIRCHE‐II numbers. Immunogenic HLA contained higher PIRCHE‐II numbers than nonimmunogenic HLA. Moreover, the probability of antibody production during pregnancy increased with the number of PIRCHE‐II. In conclusion, our data suggest that the number of PIRCHE‐II is related to the formation of child‐specific HLA antibodies during pregnancy. Present confirmation of the role of PIRCHE‐II in antibody formation outside the transplantation setting suggests the PIRCHE‐II concept is universal.     

Cryptic B Cell Response to Renal Transplantation

Transplantation reliably evokes allo‐specific B cell and T cell responses in mice. Yet, human recipients of kidney transplants with normal function usually exhibit little or no antibody specific for the transplant donor during the early weeks and months after transplantation. Indeed, the absence of antidonor antibodies is taken to reflect effective immunosuppressive therapy and to predict a favorable outcome. Whether the absence of donor‐specific antibodies reflects absence of a B cell response to the donor, tolerance to the donor or immunity masked by binding of donor‐specific antibodies to the graft is not known. To distinguish between these possibilities, we devised a novel ELISPOT, using cultured donor, recipient and third‐party fibroblasts as targets. We enumerated donor‐specific antibody‐secreting cells in the blood of nine renal allograft recipients with normal kidney function before and after transplantation. Although none of the nine subjects had detectable donor‐specific antibodies before or after transplantation, all exhibited increases in the frequency of donor‐specific antibody‐secreting cells eight weeks after transplantation. The responses were directed against the donor HLA‐class I antigens. The increase in frequency of donor‐specific antibody‐secreting cells after renal transplantation indicates that B cells respond specifically to the transplant donor more often than previously thought.     

Role of flow cytometry to define unacceptable HLA antigens in lung transplant recipients with HLA-specific antibodies

BACKGROUND: Antidonor HLA-specific antibodies have been associated with hyperacute rejection and primary graft failure in lung transplant recipients. Thus, transplant candidates with HLA-specific antibodies generally undergo prospective crossmatching to exclude donors with unacceptable HLA antigens. However, the need to perform a prospective crossmatch limits the donor pool and is associated with increased waiting list times and mortality. A virtual crossmatch strategy using flow cytometry, which enables precise determination of HLA-specific antibody specificity, was compared to prospective crossmatching in sensitized lung transplant candidates. METHODS: In all, 341 lung transplant recipients were analyzed retrospectively (April 1992 to July 2003). Sixteen patients with HLA-specific antibodies underwent transplantation based on flow cytometric determination of antibody specificity and 10 underwent prospective crossmatching. RESULTS: Freedom from bronchiolitis obliterans syndrome (BOS) at three years was similar in those undergoing a virtual crossmatch, those undergoing prospective crossmatching, and those without HLA-specific antibodies (80.4% +/- 13.4, 85.7% +/- 13.2, and 73.8% +/- 2.8, respectively, P = 0.88). Three-year survival was also comparable (87.5% +/- 8.3, 70.0% +/- 14.5, and 78.5% +/- 2.4, respectively, P = 0.31). Elimination of prospective crossmatching for sensitized patients was associated with a significant decrease in time on the waiting list (P < 0.01) and in waiting list mortality (P < 0.05). All 16 patients undergoing a virtual crossmatch had negative retrospective crossmatches. CONCLUSIONS: By carefully determining the specificity of HLA-specific antibodies, flow cytometry methodologies enable the prediction of negative crossmatch results with up to 100% accuracy, enabling the determination of appropriateness of donors. Using this virtual crossmatch strategy, crossmatching can be safely omitted prior to lung transplantation, thereby decreasing waiting list time and mortality rates for candidates with HLA-specific antibodies.