Cell Crossmatch Research Articles

Liver transplantation with a strongly positive crossmatch: Case study and literature review

A positive crossmatch has been associated with increased risk in liver transplantation. To study the clinical significance of preformed donor-specific human leukocyte antigen antibodies (DSAs) in liver transplantation, we reviewed patients who underwent liver transplantation with a strongly positive flow cytometry crossmatch. DSAs were evaluated with a Luminex solid phase assay. The complement-fixing ability of DSAs was tested with a complement component 1q (C1q) assay. Using an assay correlation between complement-dependent cytotoxicity crossmatch, flow cytometry crossmatch, and DSA results, we reviewed the effects of DSAs on the outcomes of our patients as well as reported cases in the literature. Five of 69 liver recipients had a strongly positive crossmatch: 4 had a positive T cell crossmatch [median channel shift (MCS) = 383.5 ± 38.9], and 5 had a positive B cell crossmatch (MCS = 408.8 ± 52.3). The DSAs were class I only in 1 patient, class I and II in 3 patients, and class II only in 1 patient. Cholestasis, acute rejection, or both were observed in 3 of the 4 patients with a positive T cell crossmatch with an MCS approximately greater than 300. The C1q assay was positive for 3 patients. Two had either persistent cholestasis or early acute rejection. One patient who was treated with preemptive intravenous immunoglobulin had an unremarkable outcome despite a positive C1q result. One of the 2 patients with a negative C1q assay experienced persistent cholestasis and early and recurrent acute rejection; the other had an unremarkable outcome. None of the patients died or lost a graft within the first year of transplantation. Our study suggests that human leukocyte antigen antibody screening, flow cytometry crossmatch MCS levels, DSA mean fluorescent intensity levels, and C1q assays may be useful in assessing the risk of antibody-mediated rejection and timely interventions in liver transplantation.

The outcome of the endothelial precursor cell crossmatch test in lymphocyte crossmatch positive and negative patients evaluated for living donor kidney transplantation

The presence of human leukocyte antigen (HLA) and non-HLA antibodies (Abs) in kidney transplant recipients is associated with graft rejections. This study reports the results of an endothelial precursor cell crossmatch (EPCXM) test for detection of non-HLA Abs and its correlation to lymphocyte crossmatch (LXM) test results, the degree and type of sensitization, and transplantation (Tx) outcome in patients evaluated for living donor (LD) kidney transplantation (KTx). Patients were tested before any pre-transplantation (pre-Tx) treatment and at Tx. Pre-Tx treatments included B cell depletion and Ab removal. Patient records were reviewed for assessment of renal graft function, results of biopsies, and identification of complications affecting the graft. Pre-Tx sera from 32% of the LD patients had IgG and/or IgM-binding donor EPCs. Twenty-five percent of the patients were EPCXM IgM+. Of the patients with negative LXM tests, 25% had EPC Abs mainly of IgM class not reactive with HLA. There was no difference in rejection frequency or serum creatinine levels between the EPCXM+ and EPCXM- groups. The pre-Tx EPCXM+ group had significantly more patients with delayed graft function. Prospective studies with appropriate control groups are needed to establish whether pre-treatments aiming at removing anti-endothelial cell antibodies, as detected by the EPCXM pre-Tx, have a beneficial effect on short-term and long-term graft survival.

1026-LB

To investigate the case of a potential Living Donor Renal Transplant with Negative CDC-AHG and Positive Flow cross match with diluted serum sample having high to moderate levels of multiple Class I and Class II DSAs. Both Patient and living related donor were HLAtyped for A, B, C, DR, DQ, DP by reverse SSOP-PCR.ClassI and ClassII HLA specific antibodies were determined by Luminex based single antigen bead assay. Serum samples before and after plasmapheresis/IVIG were tested for antibodies with and without OTTtreatment. One of the current serum samples was also tested for HLA-Specific IgM antibodies, and Clq binding antibodies. T and Bcell cross matches were performed on pronase treated cells by Flow cytometry using serum samples with and without OTTtreatment. The CDCAHG assay was performed on serum samples with and without OTT treatment. The patient consistently had both Class I and Class II DSAs against A74, Cw2, Cw4, DRl3, DR1S, DRS1, and DRS2 on serum samples collected in March 2012, and July 2012. After 2 rounds of plasmapheresis and IVIG treatment in the last week of July 2012, the DSA levels remained high, although the MFI values of some of the DSAs dropped insignificantly. The flow cytometry cross match was negative with untreated serum before plasmapheresis and was positive on NHS mixed serum (which is a 1:2 dilution), and in serum diluted 1:2 using PBSindicating a slight prozone effect. The CDC-AHG and CDCAmos cross matches with T and B cells were positive on untreated pre-plasmapheresis serum sample. However, after Dn treatment, the CDC-AHG and CDC-Amos cross matches were negative for both T and B cells indicating the CDC reactivity in untreated serum sample was due to IgM antibodies. It is to be noted that this same serum sample had multiple Class I and Class II DSAs detected by the Luminex SAB assay using anti-lgG as secondary antibodies. In order to determine the complement activating function of these DSAs, the pre-plasmapheresis serum sample was tested by C1qbinding assay and it turns out that there were no DSAs that reacted in the C1q binding assay. We also tested this pre-plasmapheresis serum for IgM class of HLA Class I & II antibodies, and only 4 Class I and 2 Class II antibodies were identified-none were DSAs; however the IgM class of HLA-A23 antibody could have clones that might react with the 9p public Epitope on the Donor’s HLA-A 2. And the Cw1 antibody could cross react with the donor’s Cw4 which might have contributed for the allo CDCT and B cell cross match reaction with untreated serum. Two serum samples were tested after plasmapheresis for DSAs, T and B cell flow and CDC-AHG cross match and the reaction pattern remained the almost the same except that the Flow cytometry cross match was positive on undiluted serum sample as well and CDC-AHG was positive with B cells using untreated serum, and negative when tested with Dn treated serum. In general, it appears that the patient has high levels of DSAs likely to be of the non-complement binding or weakly complement binding class of IgG antibodies namely, IgG2/4 subtypes. The Clinical implications of these indings will be discussed.

Development of De Novo Anti-HLA Antibodies in Pediatric Heart Transplant Recipients

Purpose There is increasing evidence that the de novo development of anti-HLA antibodies (Abs) following solid organ transplantation may be associated with negative outcomes including rejection in the first year and graft loss; especially de novo donor-specific antibodies (DSAs). Little data is available in paediatric heart transplant (HTx) recipients. We sought to prospectively determine the incidence, class and early impact of de novo anti-HLA Abs in a cohort of pediatric HTx recipients. Methods and Materials Following informed consent, serial panel reactive antibody (PRA) testing post-HTx (weeks 1, 2, & 4, and months 6 & 12) was done in 25 patients (14 males) transplanted between Mar 2008 & Mar 2010. Development of DSAs was correlated with rejection, allograft vasculopathy (AV) and survival. Results Median age at HTx was 8 yrs (1.5 mos – 16.3 yrs) with mean follow-up time of 15 mos (1.5 – 31 mos). 4 patients were highly sensitized pre-HTx (cPRA > 10%); 2 to Class II and 2 to both Class I & II. 4 patients had a positive crossmatch (XM), 1 T cell, 3 B cell. 18 (72%) patients developed de novo anti-HLA Abs at a median of 6 wks (IQR: 10 days – 1.7 yrs) post-HTx: anti-Class I in 6 (33%), anti-Class II in 1 (6%), and Both in 11 (61%). De novo DSAs occurred in 12/25 (48%) at a median of 2 mos (IQR: 1 month – 1.6 years) post-HTx: anti-Class I in 5 (42%), anti-Class II in 5 (42%), and Both in 2 (16%). 14 (56%) patients had evidence of acute cellular rejection (ACR) ≥ ISHLT 2 R on biopsy; 6 with multiple episodes. A positive T cell crossmatch (p Conclusions The majority of pediatric HTx recipients develop de novo anti-HLA antibodies within the first 2 years post-HTx with half being donor-specific. Anti-Class I antibodies were associated with cellular rejection but not mortality. Further study is required to determine the long term impact on graft and patient survival.

Increased Frequency of Class II HLA-DQ Donor-Specific Antibodies Is Associated with Mixed Cellular and Humoral Rejection in Lung Transplantation

Purpose The significance of donor-specific HLA antibodies (DSA) in lung transplantation (LTx) has emerged with the introduction of solid phase assays for antibody detection. Using ELISA based screening we have shown that 14% of LTx had de novo DSA and most (78%) developed chronic allograft rejection vs. 24% in patients without DSA. Further evaluation of DSA with Luminex (L) based methods for screening, specificity and functional analysis is needed to determine the impact of pre-formed and de-novo DSA on outcome. Methods and Materials Patients who were transplanted January 2011 to July 2012 and were screened prospectively for DSA with L-PRA were included in the analysis. The screening included samples pre-Tx and post LTx at 1 month and every 2 months in conjunction with surveillance and clinical bronchoscopies. The specificity of DSA and strengths (MFI) determined with single antigen beads (L-IgG) and complement binding with L-C1q- assay (L-C1q) was associated with cellular rejection and antibody mediated rejection. Results This study was done on 149 consecutive LTx with a minimum follow-up of 3 months and adequate DSA screening. All had negative CDC T and B cell crossmatches. 5% had DSA pre-Tx that persisted post-Tx and 23% had de-novo DSA within 3-6 months post LTx. Overall, 55% of LTx had high grade ACR; de-novo DSA was higher in ACR (22/82, 27%) vs. no ACR (5/67, 7%) (p 70%) de-novo DSA were HLA-DQ specific including Ab towards DQB, DQA and combination of DQB/DQA pairs. Severe ACR (n=4) with DSA and C1q reactivity was treated with plasmapheresis, IVIG and Rituximab for AMR. Patients with ACR and low level or declining DSA that had a transient or no C1q reactivity (n=19) were not treated for AMR. DSA in LTx without ACR were also low level, transient and without C1q reactivity. Conclusions The frequency of de-novo DSA is higher than previously reported. Persistent, high grade ACR with C1q binding class II HLA-DQ DSA was associated with AMR.

The Impact of Repeat HLA Mismatches on the Development of De Novo Donor Specific HLA Antbodies (DSA) in Lung ReTransplants

Purpose De Novo DSA is associated with increased risk of Bronchiolitis obliterans Syndrome (BOS) and allograft loss however the characteristics of De Novo DSA (DN DSA) that develops after repeat mismatched retransplant is unclear. We investigate the effect of repeat HLA mismatches on the development of DN DSA following lung retransplants. Methods and Materials Our study included re-transplants at our institute (n=15). We examined pre-Tx and DN DSA, as measured by single antigen Luminex beads specific to mismatched HLA antigens of the donor. Results Distribution of positive T cell flow crossmatch to the 2nd donor by pre-Tx DSA, mean number of mismatches against the 1st, 2nd donors and repeat MM are shown in table 1 . Characteristics of De novo DSA is shown in Table 2 . Twelve recipients had repeat mismatches. Five developed DN DSA after 1st donor, 2 developed DN DSA after 2nd donor (one of them within the first month) and none developed DN DSA to a repeat mismatch ( Table 2 ). Two individuals had a positive T cell crossmatch to the second donor. However, neither of these patients developed DSA after the second transplant. Conclusions Based on a realtively limited number of repeat lung transplants, it appears that repeat HLA mismatches are not associated with increased risk of development of de novo DSA to these antigens. The impact of repeat HLA mismatches on long term transplant outcomes has not been assessed in this study. These results are clinically relevant to donor selection criteria for lung retransplants and warrant further investigation in a larger cohort. Table 1 Pre-Tx DSA Mean # of 1st donor MM Mean # of 2nd donor MM Mean # of Rpt MM T Cell XM Neg (n=13) 12 (92%) 7.2 (4-11) 7.9 (6-10) 1.8 (0-4) T cell XM Pos (n=2) 1 (50%) 9.5 (7-12) 6.5 (6-7) 2.1 (1-2) Table 2 DSA to 1st Donor (n=5) DSA to 2nd Donor (n=2) DSA to Repeat MM (n=0) DSA Specificity Class I only 3 (60%) 2 (100%) 0 A23, A31, A68, B7, B45, Cw7 Class II Only 2 (40%) 0 (0%) 0 DRw53, DQ2 Class I + II 0 (0%) 0 (0%) 0 NA DSA in the first month 0 (0%) 1 (50%) 0 A31

Pronase Treatment Interfering with T Cell Crossmatch

Introduction: Pronase was used to remove Fc receptor and/or CD20 on cell surface in order to reduce non-specific reactivity of B cell flow cytometry crossmatch. However, the pronase treatment may interfere with T cells resulting in false positive T cell crossmatch. Recently, we observed three crossmatches with unexpected positive T cell reactivity. Methods: Lymphocytes isolated from either peripheral blood samples or tissues (lymph nodes or spleens) were treated with pronase prior to crossmatch. The cells were incubated with serum samples of patients. PerCP-anti-CD3 and PE-anti-CD19 conjugates were added to distinguish T and B cells. Crossmatch reactivity on cells was marked by addition of FITC-anti-human IgG conjugate. The crossmatches were analyzed by flow cytometry. Pooled positive and negative control sera were included in each crossmatch run. HLA antibody screening and identification were performed by using Flow PRA and Luminex single antigen bead assay. Results: In crossmatches of 3 recipients (R1, R2 and R3) with 3 deceased donors (D1, D2 and D3), unexpected positive T flow cytometry crossmatches were observed. All 3 recipients had no detectable HLA antibodies. The crossmatches were repeated with pronased and non-pronased D1 and D2 cells versus DTT-treated and non-DTT-treated sera of R1 and R2. A wash step following pronase treatment was added in crossmatch of D2 versus R2. In the crossmatch of D1 versus R1, a negative T cell crossmatch was obtained with non-pronased cells regardless if the serum was treated with or without DTT. Interestingly, DTT-treated serum showed a negative T cell crossmatch regardless if the cells were treated with or without pronase. In crossmatch of D2 versus R2, non-pronased cell crossmatch was negative and pronased cell crossmatch was positive regardless if the serum was treated with or with DTT and with or without a wash step. A negative T cell crossmatch was obtained for R3 when cells of D3 were not treated with pronase, while a positive T cell crossmatch was detected when cells were pronased. High dose pronase treatment resulted in stronger crossmatch reactivity for T cell and B cell as well when compared with standard dose pronase treatment. Conclusions: Our findings suggest the unexpected positive T cell crossmatches in these 3 recipients were caused by pronase treatment. While pronase treatment removed Fc receptors and/or CD20, it might have changed the conformation of other antigens on cell surface resulting in accessibility to epitopes of denatured antigens. If the recipients had antibodies against denatured antigens, a positive crossmatch might be detected. The clinical relevance of a positive crossmatch caused by antibodies to denatured antigens may not be significant.

Significance of Pretransplant False Positive Lymphocytotoxic Crossmatch in Liver Transplant Recipients

Purpose: At the time of transplantation, the recipient serum is tested with the prospective donor lymphocytes to identify specific reactivity in the donor-specific crossmatch. A positive crossmatch is a contraindication for kidney transplantation because of the higher incidence of antibody mediated rejection. However, numerous studies has found that the liver is resistant to it. We investigated the relationship between the pretransplant lymphocytotoxic crossmatch results and the long-term outcome after liver transplantation in a single center. Methods: From January 1996 to December 2010, 1021 living or deceased donor liver transplant recipients were included. Their medical records and pretransplant crossmatch results were collected. Results: 69 of 1021 (6.8%) liver transplants were performed with a positive crossmatch and their outcome was compared with the remaining 952 performed with a negative crossmatch. No significant differences in rejection, biliary complication, vascular complication, primary disease recurrence and de novo malignancy were found in negative and positive T- or B-lymphocytotoxic crossmatch recipients. Graft loss and patient survival were not inferior in the recipient group testing positive crossmatch. Besides, T and B cell crossmatch against donor showed positive results initially. But recipient's autocontrol (recipient's cell + recipient's serum) also showed similar positive results. After dithiothreitol(DTT) treatment, all previous positive result was converted to negative. So, IgM class autoantibody against recipient's own antigen was strongly suggested. We defined these subpopulation as false positive crossmatch group, accounting for 41 (4.0%) recipients. Significantly high incidences of de novo malignancies, especially lymphoid malignancy including posttransplant lymphoproliferative disorder, were observed in false positive crossmatch recipients compared to those of negative controls. (P=0.018 in overall de novo malignancy, p=0.029 in lymphoid malignancy) Conclusion: This study demonstrated that the presence of circulating IgM autoantibody in the recipient may be a risk factor for de novo malignancy, specially lymphoid malignancy. Although the precise mechanism remains unclear, immunologic factors is considered to involve in the pathogenesis of de novo malignancy.

Antibody Mediated Rejection in a Living Donor Kidney Transplant Programme

Background: Acute rejection (AR) after transplant is associated with significant reduction in graft half life. With advent of newer immunosuppressive drugs, the incidence of acute rejection has decreased worldwide, but antibody mediated rejection (AMR) remains an important cause of graft loss. We analyzed spectrum of acute AMR and their short term outcomes at our centre. Methods: We analyzed 264 patients, transplanted between Feb 2010 and Oct 2011 who had at least 3 months of follow up. Immunosuppressive protocols were- TAC+MMF+ST in 169/264 (64%) patients, steroid free (SF) protocol (Induction+TAC+MMF) in 76/264 (29%), TAC+AZA+ST in 16/264 (6%) and CYC+MMF+ST in 3 (1.2%) patients with HCV infection. In SF group, 4/76 received ATG induction and rest received basiliximab as induction. In steroid group 63/187 (23.8%) patients received basiliximab induction and 8 patients who were second or third transplant (1 patient) received ATG as induction. NIH CDC cross match was negative in all patients prior to transplantation. Tacrolimus levels were done routinely and graft biopsy was done whenever rejection was suspected. The AMR was defined according to Banff 97 classification, i.e.- presense of 2/3 criteia-.presense of donor specific antibody (DSA), C4d positivity on IF and light microscopic features of AMR. Target trough level of tacrolimus was kept between 8-12 ng/ml for first three months, 6-8 ng/ml for next 3-6 months and 4-6 ng/ml thereafter. Outcomes were evaluated in terms of acute rejection, reversal of rejection with treatment and graft or patient loss. Results: 203/264 patients (76.8%) were males. The mean age was 40.5 (±13) years. The mean duration of follow up was 290 (±110.) days. 36/264 (13.6%) developed AR, of which 31/36 (86%) were biopsy proven (BPAR). 7/264 (2.6%) had AMR (22.5% of BPAR), 6 of these were in steroid group, 5 of them developed rejection within 4-5 days and they received thymoglobulin, plasmapheresis (PP), and intravenous immunoglobulins(IV Ig), however graft nephrectomy had to be done in 3/5 as they did not respond and there was infarction in grafts on repeat biopsy. Two patients responded completely. Two of these patients had positive DSA after transplant. However one of them later on developed chronic AMR. The sixth patient in steroid group had AMR at 7 months and had incomplete response. The only patient in steroid free group developed rejection 6 months after transplant and responded completely with same regimen. This patient later on developed chest infection and died. In three patients with graft nephrectomy, one patient received multiple blood transfusions, one patient had SLE and third patient had historic B cell cross match positivity. Overall graft survival was 96.2% and patient survival was 98.1%. Conclusions: AMR remains an important cause of resistant rejections and graft loss. More sensitive cross match tecniques might help to identify the high risk patients.

55-P

With availability of specific and sensitive HLA antibody assays, the importance of donor specific antibodies (DSA) in liver transplant has grown. Patients undergoing 2nd liver transplant are at high risk of DSA. We present contrasting outcomes in 2 patients. We retrospectively reviewed the DSA in 2 liver recipients and compared them to the clinical outcomes. Pt #1: A 28 year old woman with PBC underwent a living donor liver transplant in 2006. In the next 4 years she received her 2nd, 3rd and 4th liver transplants due to graft failures. Plasmapheresis and IVIG were used at the 3rd & 4th transplants due to high PRA. A positive B cell flow crossmatch was noted at the last transplant. DSA monitoring was initiated after the 4th transplant. Elevated levels of class II antibodies to DR4, DR53 and DQ8 persisted. Patient had recurrence of jaundice after 6 months and showed short lived response to thymoglobulin, plasmapheresis, IVIG and Rituximab with rebound of DSA within a month of antibody therapy. Coincidentally, donors 2, 3 and 4 all had DR4, DR53 and DQ8. The patient underwent Bortezomib therapy as liver biopsy continued to show mixed portal infiltrate & bile duct injury. Pt #2: A 25 yr old woman with autoimmune hepatitis underwent liver transplant in 2006. Graft function deteriorated following steroid non-responsive rejection treated with Thymoglobulin & OK T3. Retransplant was done in early 2011. B cell crossmatch was positive and patient received thymoglobulin, plasmapheresis and IVIG. DSA assay showed the presence of anti-A2, -A11, -DR53, and -DQ8. During a follow up of 11 months, graft function was excellent despite the persistence of high titers of Class II antibodies. In contrast, level of class I antibodies remain under cutoff. Sensitization status needs serious consideration in retransplantation of liver. Avoidance of donors with unacceptable antigens may be crucial in some liver retransplant recipients. A positive B cell crossmatch may portend a poor outcome.

180-P

Aim Single antigen bead (SAB) assay allows in-depth characterization of HLA antibody specificities. In addition to detection of antibodies against natural HLA antigens, the SAB may also detect antibodies against denatured HLA antigens. Recently, we observed antibody reactivity to all DR antigens (pan-DR) in serum samples of two transplant candidates using the Luminex SAB assay. This study was to characterize the pan-DR antibodies detected by Luminex SAB on two serum samples. Methods Serum samples received from two transplant candidates were analyzed in the study. Initial antibody screening and specificity tests were performed using Flow PRA and Luminex SAB. Additional tests using Flow SAB, Flow phenotype bead assay and Flow T and B cell crossmatch were performed to confirm the presence of HLA antibodies. Results Flow PRA was clearly negative for both samples, while Luminex SAB surprisingly revealed antibody reactivity to all DR antigens. No antibodies to DQ and DP were detected. However, no HLA antibodies were detected by Flow phenotype bead assay. In the Flow SAB assay, patient #1 showed reactivity with majority of the DR antigen beads, while patient #2 showed no any binding reactivity. The crossmatch with a donor cell carrying DR4, DR15, DR51, and DR53 was completely negative for both serum samples. No specific antibody removals were observed on serum samples adsorbed using the donor cells in a Luminex SAB assay. Conclusions Flow PRA and Flow phenotype beads are coated with natural HLA antigens extracted from cell lines, while SABs are coated with recombinant HLA antigens. The antibody binding detected by SAB, but not by FlowPRA and Flow phenotype, may indicate the presence of antibodies against denatured antigens. Our results suggest that the pan-DR antibodies detected in these patients were possibly antibodies against denatured HLA antigens, more likely to a shared epitope uncovered on denatured DR antigens. Antibodies to denatured antigens would not result in crossmatch reactivity.

190-P

Aim Crossmatch (XM) for deceased solid organ transplant is a critical and challenging process due to limited cold ischemia time, the need for adequate cell yield and purity of cell isolation, and the process must be quick. In this study we evaluated different cell separation methods to select the most efficient one for clinical transplant crossmatch. Methods Lymphocytes were isolated following lab procedure and/or manufacturer instructions (STEMCELL Technologies Inc.) using Ficoll, RosetteSep, RosetteSep with SepMate Tube, EasySep magnetic selections. Flow T and B cell crossmatches were performed following lab procedure. Results The purity, yield, and average time spent isolating lymphocytes from PB samples obtained from 6 volunteers are given in the following table ( Table 1 ). Conclusions EasySep custom positive selection delivered excellent yield and purity; however, the cells showed strong background binding in the flow XM, making them unacceptable for use in the flow XM. The Ficoll method gave the highest yield with the lowest purity, which may require additional centrifugations to remove red cells, platelets and granulocyte, thus adding time for isolation. Many lymphocytes were lost during the separation using the RosetteSep and RosetteSep with SepMate Tube. However, the methods offer excellent cell purity and no additional separation is needed. Overall, the use of RosetteSep with SepMate tubes could be a practical method for routine lymphocyte isolation for deceased solid organ transplantation.

53-P

Aim The presence of antibodies against human leukocyte antigen (HLA) is a major challenge for successful kidney transplantation and results in prolonged waiting times. The aim of the study is to investigate the efficacy of high dose intravenous immune globulin (IVIG) on HLA antibody reduction and graft dysfunction in renal transplant recipients transplanted against DSA. Methods Between 07/2005 and 04/2012, 53 sensitized patients whose T and B flow crossmatches were under 200 median channel shift were transplanted with single or multiple dose of IVIG (2 g/kg). Levels of HLA antibodies were measured using single antigen Luminex assay, T/B cell crossmatches were performed by cytotoxicity and flow cytometry. Results All 53 patients displayed DSA to their donors pretransplant. Among them, 25/53 (47%) patients had a calculated PRA (cPRA) >90%. 26 patients had pretransplant DSA to HLA class I, 17 had DSA to class II and 10 had DSA to both class I and class II. 10 (18.8%) patients experienced antibody mediated rejection (AMR) posttransplant, and 5 (9.4%) patients were diagnosed with acute cellular rejection (ACR). 65% (17/26) of patients transplanted with pretransplant DSA to only class I antigens lost their DSA after transplant. However, for patients with pretransplant DSA to class II, only 5/17 (29%) patients showed no detectable DSA posttransplant (p = 0.02). For the 10 patients who had pretransplant DSA to both class I and class II, 4 patients no longer had detectable DSA posttransplant. Four patients had persistent class II DSA and 2 patients had persistent DSA to both class I and class II posttransplant. Conclusions IVIG seems to be more efficient in removing HLA class I antibodies than class II. Patients who had pretransplant DSA to HLA class II had higher risk of AMR compared to patients who had DSA to class I, with the highest risk in patients with DSA to both. Additional study should be performed to understand the differential mechanisms of IVIG on HLA class I and class II antibodies.

39-P

Aim Low level antibodies may not always produce a positive flow crossmatch and increase the risk for transplant. The aim of this study was to investigate the impact of increasing the MFI cutoff for listing the unacceptable antigens (UA) in UNET. UA are use in virtual crossmatches and exlude recipients without performing crossmatches. Methods Antibody detection was performed by One Lambda LABScreen Single Antigen HLA Class I and II beads. The normalized MFI was used to calculate the cutoff. The MFI cutoff for a positive result was increased from 1,500 to 3,000. T and B cell crossmatches were performed by flow cytometry with pronase treated cells and reported as MCS. The T cell cutoff for positive was 61 and B cell was 93 which is 3 standard deviations above our negative control serum. The ten highest ranking recipients on the kidney runs from the first 8 deceased donor workups following the change were studied. Results Fifty six of the 126 actively listed patients from CTHH had UA entered in UNET. Low level UA were removed from 50/56 recipients. After the modification, eight recipients fell below and 21 stayed above 80% CPRA. The remainder had a CPRA range from 5-73%. 48 of the top 80 recipients on recent kidney match runs were from CTHH and further studied. 14/48 (29%) had low level antibodies present. 3/14 (21%) were flow T and B cell negative and the DSA was to DQ7 in all three crossmatches. Eleven were T and/or B cell positive. Four of the positive crossmatches were due to Cw, DQA, or DP antibodies which are not reported as UA. Three were due to low levels of multiple antibodies producing a cumulative effect. One was due to Bw6 antibody that reacted with all Bw6 beads on the Luminex assay but reacted below 3,000 MFI. The remaining four positive crossmtaches were due to historically higher MFI values. Conclusions Low level antibodies should not be added as unacceptable antigens and automatically rule out a recipient for kidney transplantation. Some crossmatches will be negative.

4-OR

To elaborate the importance of HLA-DPB1 typing in Pre-transplant solid organ candidates. To show that HLA-DPB1 typing on Deceased Donors can enhance the virtual crossmatch system, especially with highly sensitized pre-transplant candidates. Currently UNOS does not require or accept HLA-DBP1 typing. This excludes HLA-DPB1 from the virtual crossmatch algorithm. HLA-DPB1 can cause antibody-mediated rejection. Molecular typing performed for HLA-A, B, C, DQB1, and DPB1 using SSP (Olerup). Antibody tests were performed with LabScreen Single antigen beads Class I and II beads (One Lambda) on serum samples treated with DTT. Reviewed highly sensitized renal pre and post transplant cases involving HLA-DPB1 antibodies. Case 1: Highly sensitized 65 yr female renal candidate with a cPRA of 100%, received a transplant with a deceased donor in October 2010. Donor typing was considered a zero mismatch. Early antibody mediated rejection was suspected due to an increase in serum creatinine and decrease in urine output. Anti-HLA antibody specificity analysis revealed strong HLA-DPB1 antibodies. Molecular typing for HLA-DPB1 was performed [ Table 1 ]. DSA performed two days post transplant showed the HLA-DPB1 ∗ 0101 at 4900 MFI. By the tenth day post transplant HLA-DPB1 ∗ 0101 was 17,100 MFI. The kidney had stopped functioning and showed intense C4d deposition. Case 2: Highly sensitized 56 yr male renal candidate active on the waitlist having had three previous transplants and a current cPRA of 91%. Since he was listed in May 2011 he has had several deceased donor offers. However, Single Antigen Beads revealed multiple HLA-DPB1 antibodies leading to both positive flow cell and virtual crossmatches. It is now our protocol to perform molecular HLA-DPB1 typing on deceased donors, as well as, potential recipients. The more complete a donor’s HLA typing the more we are able to assign DSA (post-transplant) and help in the diagnosis and treatment.

Detection of complement‐fixing and non‐fixing antibodies specific for endothelial precursor cells and lymphocytes using flow cytometry

Donor human leukocyte antigen (HLA)-specific antibodies (Abs) with the ability to activate complement are associated with an increased risk of early Ab-mediated rejection (AMR) of kidney allografts. In recent years, also non-HLA Abs-binding endothelial cells have been shown to elicit early AMR. Donor-specific anti-endothelial cell Abs escape detection in the pre-transplant evaluation if only lymphocytes are used as target cells in crossmatch tests. We addressed whether endothelial precursor cells (EPCs) could be used for detection of complement-fixing as well as non-fixing Abs and if complement factor and immunoglobulin G (IgG) deposition on co-purified T and B cells correlated to the outcome of the T- and B-cell complement-dependent cytotoxicity assay. Deposition of complement factors C3c and C3d, but not C1q nor C4d, were detected on EPCs and lymphocytes upon incubation with HLA Ab-positive sera. There was a correlation between the amount of C3c deposition and IgG binding on EPCs (R(2) = 0.71, P = 0.0012) and T cells (R(2) = 0.74, P = 0.0006) but not for B cells (R(2) = 0.34, P = 0.059). The specificity and sensitivity for C3d deposition on endothelial precursor cell crossmatch (EPCXM) T cells vs the T complement-dependent cytotoxicity (CDC) assay were 69% and 72%, respectively. The EPCXM B-cell C3d assay had considerably lower sensitivity (39%) than the B CDC assay. Altogether, this novel assay based on the detection of complements factors on EPCs and lymphocytes by flow cytometry may widen the diagnostic repertoire and thereby improve the clinical management of patients undergoing kidney transplantation.

Implications of a positive crossmatch in liver transplantation: A 20-year review

Whether a positive crossmatch result has any relevance to liver transplantation (LT) outcomes remains controversial. We assessed the impact of a positive crossmatch result on patient and graft survival and posttransplant complications. During a 20-year period, 2723 LT procedures with crossmatch results were identified: 2479 primary transplants and 244 retransplants. The rates of positive B cell and T cell crossmatches were 10.1% and 7.4%, respectively, for primary transplants and 14.6% and 6.4%, respectively, for retransplants (P = 0.049 for a B cell crossmatch). Across all primary transplants, females (P < 0.001) and patients with autoimmune hepatitis (P < 0.001) had greater frequencies of positive crossmatches. There was no effect from race or age. For both primary transplants and retransplants, patient survival and graft survival were not affected by the presence of a positive crossmatch. With respect to posttransplant complications, there were no differences in rejection episodes (hyperacute, acute, or chronic) or technical complications (biliary and vascular) between negative and positive crossmatch groups. However, there were significant differences in the pathological findings of preservation injury (PI) on liver biopsy samples taken at the time of transplantation and within the first week of transplantation (P = 0.003 for B cells and P = 0.03 for T cells). In summary, a positive crossmatch had no significant impact on patient survival or graft outcomes. However, there was a significantly higher incidence of PI in primary LT recipients with a positive crossmatch. This finding is important for a broader understanding of PI, which may include a significant immunological component.

5-P Optimized pronase treatment eliminates false positive B cell crossmatch caused by desensitization protocol with rituximab: High dose of pronase treatment affects CD19 expression

5-P Optimized pronase treatment eliminates false positive B cell crossmatch caused by desensitization protocol with rituximab: High dose of pronase treatment affects CD19 expression

De novo development of circulating anti-endothelial cell antibodies rather than pre-existing antibodies is associated with post-transplant allograft rejection

Anti-endothelial cell antibodies (AECAs) are thought to be involved in the development of renal allograft rejection. To explore this further, we determine whether AECAs play a role both in predicting the incidence of allograft rejection and long-term outcomes by analysis of serum samples from 226 renal allograft recipients for AECAs pre- and post-transplant. Surprisingly, the presence of pre-existing AECAs was not associated with either an increased risk of rejection or a detrimental impact on recipient/graft survival. Subsequent de novo AECAs, however, were associated with a significantly increased risk of early acute rejection. Moreover, these rejections tended to be more severe with a significantly increased incidence of both steroid-resistant and multiple episodes of acute rejection. The acute rejections associated with de novo AECAs did not correlate with C4d deposition at the time of renal biopsy, but did demonstrate an association with the presence of glomerulitis and peritubular capillary inflammation. Significantly more patients with de novo AECAs developed graft dysfunction. Thus, our prospective study suggests the emergence of de novo AECAs is associated with transplant rejection that may lead to allograft dysfunction.

Limitations of Rituximab/IVIg desensitization protocol in kidney transplantation; Is this better than a tincture of time?

A plasmapheresis-free protocol for desensitization of donor kidney transplant candidates with high Calculated Panel Reactive Antibody (CPRA) was initiated at our center. The protocol was adopted from previously published work by Vo, Jordan et al. Five patients with CPRA of 94 ± 18%, awaiting kidney transplant from living or deceased donors received rituximab (1 g × 2 doses) and intravenous immunoglobulin (IVIG 2 g/kg × 2 doses) without plasmapheresis. Levels of donor specific antibodies (DSA) and T/B cell crossmatches were followed using solid phase flow cytometry. Three out of 5 patients were sensitized only to Class II HLA antigens. All patients had very high levels of alloantibodies before initiation of the treatment. All of the candidates initially demonstrated reduced levels of HLA antibody, but statistical significance was only obtained in one patient Class II antibody and in another only for Class I. Depletion was transient with observed antibody rebound. Rituximab effectively depleted CD20 cells in peripheral blood. None of the patients were transplanted due to persistently high levels of antibody and strong positive flow cytometry crossmatches. Under this protocol, reduction of HLA antibodies in patients with high levels was insufficient. Highly allo-sensitized patients with a CPRA above 85% may not benefit from a combination of rituximab-IVIG alone. The previously published protocol does not help all patients achieve an acceptable crossmatch. An individualized approach to the treatment of highly sensitized patients is still required.