Unacceptable HLA Antigens Research Articles

HLA sensitization is associated with an increased risk of primary graft dysfunction after heart transplantation

Consecutive adult HT recipients (n=596) from 1/2015to12/2019 at 2 US centers were included. Severity of PGD was based on the 2014 International Society for Heart and Lung Transplantationconsensus statement. For each recipient, unacceptable HLA antigens were obtained and locus-specific cPRA (cPRA-LS) and pre-HT donor-specific antibodies (DSA) were assessed. Univariable logistic modeling showed that peak cPRA-LS for all loci and HLA-A was associated with increased severity of PGD as an ordinal variable (all loci: OR 1.78, 95% CI: 1.01-1.14, p=0.025, HLA-A: OR 1.14, 95% CI: 1.03-1.26, p=0.011). Multivariable analysis showed peak cPRA-LS for HLA-A, recipient beta-blocker use, total ischemic time, donor age, prior cardiac surgery, and United Network for Organ Sharing status 1 or 2 were associated with increased severity of PGD. The presence of DSA to HLA-B was associated with trend toward increased risk of mild-to-moderate PGD (OR 2.56, 95% CI: 0.99-6.63, p=0.053), but DSA to other HLA loci was not associated with PGD. Sensitization for all HLA loci, and specifically HLA-A, is associated with an increased severity of PGD. These factors should be included in pre-HT risk stratificationto minimize the risk of PGD.

Pre-Formed Donor-Specific Antibodies Impact Long Term Survival of Heart Transplants

We previously reported that heart transplant (HTx) recipients with pre-formed, anti-HLA antibodies at low titer had one-year survival that was comparable to patients without any anti-HLA antibodies. As a result, we defined unacceptable HLA antigens by MFI>1000 in serum at 1:16 dilution. The current study evaluates how pre-formed, low-titer, donor-specific antibodies (DSA) and positive crossmatch affect long term survival of HTx recipients. Total 495 adult recipients transplanted at our center between Jan. 2007 and Dec. 2016 were studied. Prior to HTx, recipients had HLA antibody screening (FlowPRA) and specificity testing (LABScreen). Unacceptable HLA antigens were defined by MFI>1000 in serum at 1:16 dilution. Acceptable donors were selected by virtual crossmatch using the criteria above, and flow cytometry crossmatch (FCXM) was performed peri- or post-HTx. Recipients transplanted across low titer DSA, positive in undiluted serum but negative at 1:16 dilution, received peri-operative plasmapheresis. We used log-rank testing to evaluate survival in the following groups compared to their controls: (1) presence of any pre-formed HLA antibodies; (2) presence of pre-formed DSA; and (3) positive FCXM. Of 495 HTx recipients, 179 (36%) had pre-existing HLA antibodies, and their overall survival was not different from patients without HLA antibodies (p=0.883). However, median survival time was reduced in patients with pre-formed DSA (n=62), compared to DSA-negative recipients (n=117): 9.1 years (95% CI 3.52 to 14.65) vs. 10.9 years (95% CI 6.13 to 15.70); p=0.02. The survival rates for 1-, 3-, 5-year of DSA-negative recipients were 91.5%, 85.4% and 79.5%, and the DSA-positive recipients were 83.9%, 66.1% and 61.7%, respectively. Total 62 recipients had pre-HTx DSA. Among them, 26 had a positive FCXM (46%). The survival rates of FCXM-positive cases were lower than FCXM-negative cases (n=36) at 1, 3, and 5 years: 80.8%, 57.9%, 52.1% vs. 86.1%, 71.4%, 67.6%. Our findings suggest that pre-formed DSA, even at low titer, negatively impacted HTx survival. Cases with positive FXCM had the worst outcomes. Avoiding all pre-formed DSA may improve survival for HTx patients, but may increase waitlist mortality. To improve the HTx survival, close post-transplant monitoring and proactive immunological management may be required when pre-formed DSA are present.

P031 Unacceptable antigen listing should not be based on luminex single antigen testing only

Unacceptable HLA antigens are largely determined by testing sera with Luminex single antigen (LSA) beads. Table 1 shows the MFI values for the five DQB7 beads in three pre-transplant specimens performed on a patient with a cPRA of 99%. DQA1*05 and DQA1*06:01, but not DQB7, were listed as unacceptable antigens. A deceased donor with DQA1*01,03 and DQB5,7 became available and was virtual and flow cytometry crossmatch compatible. On post-op day 14, the patient presented with graft dysfunction. A biopsy contained evidence of antibody mediated rejection (AMR), which was supported by microarray. A donor specific antibody (DSA) screen revealed a greater than 10-fold increase in the previously negative DQB7 beads (DQA1*02:01/DQB1*03:01 and DQA1*03:01/DQB1*03:01) in addition to the previously positive DQA1*05:03, DQA1*05:05, and DQA1*06:01 beads and was consistent with a DSA to DQB7. C1q testing was positive for DQA1*05 only, which was not donor specific. The patient was aggressively treated for AMR but a repeat biopsy on post-op day 25 contained a thrombotic microangiopathy consistent with AMR. The allograft was removed on post-op day 75 due to non-function. Investigation into this case revealed a previous failed transplant with a DQB7 positive donor at another institution, which was unknown to our laboratory. Although the DQA1* typing was not available for the first donor, DR/DQB linkage analysis favors DQA1*05 consistent with the pre-transplant LSA and post-transplant C1q results. Based upon the clinical course, we hypothesize DQB7 specific memory B cells, formed in response to the first donor, facilitated accelerated AMR, which may have been avoided by listing DQB7 as an unacceptable antigen. In conclusion, the results of the LSA assay are only one component of the patient’s immunological profile. Decisions regarding the listing of unacceptable antigens must additionally take into consideration previous immunizing events, like a failed transplant, and this information must be provided to the histocompatibility laboratory for optimal patient care.

HLA Population Genetics in Solid Organ Transplantation.

HLAs are fundamental to the adaptive immune response and play critical roles in the cellular and humoral response in solid organ transplantation. The genes encoding HLA proteins are the most polymorphic within the human genome, with thousands of different allelic variants known within the population. Application of the principles of population genetics to the HLA genes has resulted in the development of a numeric metric, the calculated panel-reactive antibody (CPRA) that predicts the likelihood of a positive crossmatch as a function of a transplant candidate's unacceptable HLA antigens. The CPRA is an indispensible measure of access to transplantation for sensitized candidates and is used as the official measure of sensitization for allocation of points in the US Kidney Allocation System and Eurotransplant. Here, we review HLA population genetics and detail the mathematical basis of the CPRA. An understanding of these principles by transplant clinicians will lay the foundation for continued innovation in the care of sensitized patients.

P062 Characteristics of pre-formed donor specific anti-HLA antibodies associated with their clearance or persistance

Aim Pre-formed non-cytotoxic donor specific anti-HLA antibodies (DSA) are not a contraindication for kidney transplantation. Such DSA are often cleared post-transplant, having minimal effect on graft function. However, pre-formed DSA can also persist post-transplant, potentially increasing the risk of antibody mediated injury. The aim of this study was to evaluate the characteristics of pre-formed donor specific HLA antibodies (DSA) in relation to their clearance or persistence after transplantation in a large population of kidney allograft recipients. Methods Our study population included 681 patients transplanted at our center from Jan 1st 2009 to Dec 31st 2014. Typing of HLA-A, B, C, DR and DQB genes was performed by DNA methods. Serum anti-HLA antibodies were tested at defined intervals pre- and post-transplantation using Single Antigen Beads (One Lambda/ThermoFisher). Unacceptable HLA antigens were reported to UNET using MFI cutoff values of 5000 (HLA-A, B and DR) and 10,000 (C and DQ). Prospective crossmatches were performed in all cases. Patients were transplanted based on a negative flow cytometry and/or CDC crossmatch. Results Out of 681 recipients, 108 (16%) had pre-formed DSA. At one year post-transplant, DSA were cleared in 67 patients and persisted in 41 patients. Patient age, gender and race, transplant type (live or deceased donor) and the number of DSA per patient did not significantly differ in patients who cleared versus patients with persistent DSA. However, patients with persistent DSA were more likely to have DSA directed to the donor HLA-DQB rather than HLA-A, B, C or DR antigens (p = 0.041; Fig. 1), DSA with higher MFI (p Download : Download high-res image (77KB) Download : Download full-size image Conclusions This study identifies the characteristics of preformed DSA that render them more likely to persist post-transplantation in our patient population. These findings may improve risk stratification of kidney transplant candidates with preformed DSA.

P085 Lung re-transplant with repeated mismatch HLA antigens

Avoidance of previous donor mismatched HLA antigens is often considered for lung re-transplant due to concerns of potential HLA sensitization and memory responses. However, this approach may result in the exclusion of a large number of potential donors from consideration. Recently, we successfully performed a re-transplant with a history of multiple repeated mismatches. A 30+ year old male with a past medical history of cystic fibrosis underwent two previous bilateral lung transplants in August 2004 and in November 2010. Both allografts failed due to CLAD/BOS that developed after respiratory viral infections. The patient also subsequently developed detectable class I and II HLA antibodies with a cPRA > 88% in May 2016. Among these antibodies, strong anti-A1, B7 and DQ2 were first donor HLA specific. The second donor shared an A3-B18-DR4 haplotype with the first donor but not the recipient. No HLA antibodies specific to this A3-B18-DR4 haplotype antigens or to any other mismatched HLA antigens of the second donor were detected. A third lung transplant became urgently necessary in October 2016. Adding his previous donors’ mismatched HLA antigens to the unacceptable HLA list resulted in a cPRA > 96%. Therefore, a personalized listing strategy was developed based on discussions between the HLA lab and the transplant program. A deceased donor became available in November 2016. The virtual crossmatch revealed that the donor had no unacceptable HLA antigens based on HLA antibody detection. However, this donor carried the A3-B18-DR4 haplotype which was a repeated mismatch haplotype of the previous two donors. Based on a risk and benefit assessment, this donor was accepted as the source of lungs for the third transplant. The patient received 5 plasmapheresis sessions post-transplant to prevent antibody mediated rejection. Frequent post-transplant HLA antibody monitoring has not detected development of donor specific antibodies to the third donor and the new allograft functions well. Lung re-transplant can be performed with a cross of a repeated HLA mismatch provided that the recipient has no detectable HLA antibodies specific to the current donor. Our preliminary findings obtained from our study conducted on UNOS lung transplant data supports this conclusion. An assessment of risk and benefit of crossing the repeated mismatch HLA antigens should be done on a case-by-case basis.

Meaning of HLA Antibodies Frequently Detected by Single Antigen Bead Assay

Introduction: Single Antigen Bead (SAB) assay to detect HLA antibodies is the highly sensitive and specific technique. Nowadays, almost patients awaiting a transplantation performed SAB assay to determine unacceptable HLA antigens, and waiting time for transplantation from deceased donors was considered. Because of inherent limitation of SAB kits, which use recombinant technology, false reactivity to denatured antigens could happen. False reactivity affects donor selection and waiting transplantation time. We investigated SAB data to examine false reactive results. Methods: We tested 155 pre-transplanted and 45 post-transplanted sera for anti-HLA antibodies using panel kit and SAB kit (LABScreen Luminex kits, One Lambda, Canoga Park, CA, USA). Negative Control Beads (NC) value was below 500 mean fluorescent intensity (MFI) and Positive Control Beads (PC) value was over 5000 MFI in all results. Results: In panel kit analysis, 27% of patients were positive for HLA antibodies. Sixty-four percent of patients were positive at least one HLA antibodies on SAB assay even negative in panel kit analysis. MFI of detected HLA antibodies, which were from sera for negative in panel kit and positive in SAB kit, was under 3000 in 70.9% and between 3000 and 10000 in 25.6% in Class I SAB assay, and under 3000 in 80.7% and between 3000 and 10000 in 17.1% in Class II SAB assay. Comparison with allele frequencies from our laboratory HLA typing (n = 785), three-times high frequent HLA antibodies were HLA-A*11:02 (6%), A*80:01 (4.5%), B*15:12 (10.5%), C*17:01 (18.5%), C*18:02 (4.5%), DRB1*04:04 (6.5%), DRB1*16:01 (6.5%), DRB1*16:02 (5.0%), DQ2 (A1*05:01/B*02:01; 15.5%), DQ7 (A1*05:03/B1*03:01; 6.5%, A1*05:05/B1*03:01; 7.5%, A1*06:01/B1*03:01; 4.5%), DQ8 (A1*03:02/B1*03:02; 7.5%), DQ9 (A1*03:02/B1*03:03; 6.5%), DP1 (A1*01:03/B1*01:01; 8.5%, A1*02:01/B1*01:01; 5.0%), and DP5 (A1*02:02/B1*05:01; 7.5%). Conclusion: HLA antibody with higher detection frequency than allele frequency of population was considered false reactive antibody, because a possibility of exposure would be very low. Most of false reactive HLA antibodies showed under 3000 MFI. Erroneous interpretation about HLA antibodies for patients awaiting transplantation impact on donor selection and waiting time calculation. Studying for frequently seen false reactive HLA antibody could help relevant SAB results.

Corrected Panel-Reactive Antibody Positivity Rates for Hypersensitized Patients in Turkish Population With Calculated Panel-Reactive Antibody Software

IntroductionHigh rates of panel-reactive antibody (PRA) may decrease the chance of kidney transplantation and may result in long waiting periods before transplantation. The calculated PRA (cPRA) is performed based on unacceptable HLA antigens. These antigens are identified by a program that was created based on the antibodies that developed against the HLA antigens circulating in serum and on the risk of binding of these antibodies to antigens. The antigen profile of the population and antigen frequencies can be measured, and more realistic cPRA positivity rates may be obtained using this method. Materials and MethodsWe developed a program based on the HLA antigens of 494 blood donors in 2 European Federation for Immunogenetics–accredited Tissue Typing Laboratories in Turkey. Next-generation sequencing–based tissue typing (HLA-A, -B, -C, -DR, -DQ, 4 digits) of the samples was performed. The PRA screening test was performed on 380 patients who were waiting for organ transplant from a cadaver in Istanbul Faculty of Medicine. The single antigen bead assay testing was performed to identify the antibody profiles on 48 hypersensitized patients. ResultsThe PRA testing results using the current methods were 44.6% ± 18.5%, and the cPRA rate was 86.2% ± 5.1%. The mean PRA positivity of the sensitized patients using the current methods was 44.6%; however, the rate was 86.2% using the cPRA. DiscussioncPRA shows the rate of the rejected donors according to all unacceptable antigens. The need for a list of unacceptable antigens in place of the PRA positivity rate is a real change in the sensitization-dependent calculation as cPRA positivity rate. ConclusionIn principal, implementation of cPRA will encourage many centers and laboratories to adopt a standard measurement of sensitization in Turkey. It will increase the chances of better donor match, particularly for hypersensitized patients, by the creation of an unacceptable mismatch program using cPRA software.

Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs

Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation.

P003 Impact of the new kidney allocation system on the transplant rates of highly sensitized patients. A single center experience

Aim Increasing transplant opportunities for difficult-to-match patients was a primary goal of the new kidney allocation system (KAS) implemented on Dec 4, 2014. In this study, we evaluated the impact of KAS on the transplantation rate of sensitized candidates at our center. Methods During Jan 1–Dec 4, 2014 (pre-KAS interval) and Jan 1–Dec 4, 2015 (post-KAS interval), unacceptable HLA antigens for kidney transplant candidates were reported to UNET using the following cutoffs: 5000 MFI for HLA-A, -B and -DR, and 10,000 MFI for HLA-C and -DQ antigens. Deceased donor crossmatches (XMs) were performed by flow cytometry and complement dependent cytotoxicity (CDC). Patients were transplanted based on a negative flow and/or negative CDC XM. Results A total of 613 and 1188 deceased donor XMs were performed during pre- and post-KAS intervals, respectively. The number of XMs performed for sensitized patients (cPRA>0%) increased from 116 (19%) XMs performed pre-KAS to 309 (26%) post-KAS (p 98%, 50–79% and 1–49% (Table 1). During pre- and post-KAS intervals, a total of 115 and 129 patients, respectively, received deceased donor kidney transplantation at our center. As indicated in Table 1, the number of sensitized recipients increased from 16 (14% of pre-KAS transplants) to 32 (25% of post-KAS transplants). The highest increase was seen in patients with cPRA>98% (from 0% to 8%). Transplant outcomes were analyzed in the non-sensitized (N = 97) and sensitized (N = 32) patient groups, who received deceased donor kidney allografts post-KAS. Graft survival, patient survival, creatinine levels and incidence of acute rejection were similar in the two groups. Conclusion The new KAS facilitated a marked increase in the transplant rate of sensitized candidates at our center. The HLA laboratory played a key role in achieving this goal by providing thorough testing and ensuring safe transplantation of difficult-to-match patients. Download : Download high-res image (140KB) Download : Download full-size image

Evaluation of Changes in New Calculated Panel Reactive Antibody Adopting HLA-Cw, DR51/52/53, and DQ Antigens in Koreans

BackgroundCalculated panel reactive antibody (cPRA) (%) is percentage of donors that would be incompatible with the candidate, based on the candidate's unacceptable HLA antigens. cPRA based on antigen frequencies of HLA-A, B, and DR has been used in Korea. We developed new cPRA including HLA-Cw, DR51/52/53, and DQ. Changes in new-cPRA were evaluated. MethodsWe analyzed the differences between cPRA based on HLA-A, -B, and -DR antigens (old-cPRA) from cPRA based on HLA-A, -B, -Cw, -DR, -DR51/52/53, and -DQ antigens (new-cPRA) on 125 waitlisted candidates for renal transplantation in Seoul National University Hospital. cPRA for unacceptable antigens was calculated according to 3 different cut-off values (MFI <1000, 3000, and 10000 for cPRAw, cPRAm, and cPRAs, respectively). ResultsFor HLA class I, cPRAw and cPRAm were significantly increased in new-cPRA compared to old-cPRA (median 78.3% vs 71.7%, P < .001; 34.0% vs 23.5%, P = .029, respectively). For HLA class II, cPRAw, cPRAm, and cPRAs were significantly increased in new-cPRA compared to old-cPRA (median 86.8% vs 42.6%; 58.0% vs 0.0%; 0.0% vs 0.0%, P < .001 for all). ConclusionscPRA (%) including HLA-Cw, -DR51/52/53, and -DQ showed remarkable increase, especially in HLA class II antigens. The meaning of this should be carefully interpreted through further studies considering clinical outcomes.

Allocating Deceased Donor Kidneys to Candidates with High Panel-Reactive Antibodies.

In December of 2014, the Organ Procurement and Transplant Network implemented a new Kidney Allocation System (KAS) for deceased donor transplant, with increased priority for highly sensitized candidates (calculated panel-reactive antibody [cPRA] >99%). We used a modified version of the new KAS to address issues of access and equity for these candidates. In a simulation, 10,988 deceased donor kidneys transplanted into waitlisted recipients in 2010 were instead allocated to candidates with cPRA≥80% (n=18,004). Each candidate's unacceptable donor HLA antigens had been entered into the allocation system by the transplant center. In simulated match runs, kidneys were allocated sequentially to adult ABO identical or permissible candidates with cPRA 100%, 99%, 98%, etc. to 80%. Allocations were restricted to donor/recipient pairs with negative virtual crossmatches. The simulation indicated that 2111 of 10,988 kidneys (19.2%) would have been allocated to patients with cPRA 100% versus 74 of 10,988 (0.7%) that were actually transplanted. Of cPRA 100% candidates, 74% were predicted to be compatible with an average of six deceased donors; the remaining 26% seemed to be incompatible with every deceased donor organ that entered the system. Of kidneys actually allocated to cPRA 100% candidates in 2010, 66% (49 of 74) were six-antigen HLA matched/zero-antigen mismatched (HLA-A, -B, and -DR) with their recipients versus only 11% (237 of 2111) in the simulation. The simulation predicted that 10,356 of 14,433 (72%) candidates with cPRA 90%-100% could be allocated an organ compared with 7.3% who actually underwent transplant. Data in this simulation are consistent with early results of the new KAS; specifically, nearly 20% of deceased donor kidneys were (virtually) compatible with cPRA 100% candidates. Although most of these candidates were predicted to be compatible with multiple donors, approximately one-quarter are unlikely to receive a single offer.

LBP29: TRANSPLANTATION OF SENSITIZED HEART PATIENTS

In 2010, at ASHI, we presented our preliminary data of using antibody titration to determine unacceptable HLA antigens (UAG) for selection of deceased donors for heart transplant. The one-year survival rate of recipients who had “low titer” donor-specific antibody (DSA) was comparable to recipients who had no DSA or no pre-transplant sensitization. This approach has been continuously used at our transplant center. This study was conducted to evaluate the survival of recipients who received a heart transplant between 2010 and 2013 and to assess the risk of “low titer” DSA in the current cohort. All patients that received heart transplants from January 1, 2010 and December 31, 2013 were included in this study. Prior to listing, all patients were evaluated for HLA sensitization which was defined by presence of any HLA antibodies detected by Flow antibody screening and Luminex single antigen bead assay. For sensitized recipients, a titration test at 1:16 dilution was performed. Utilizing the principle of virtual crossmatch (VXM), only donors without UAGs determined at 1:16 dilution were selected for sensitized patients. All final XMs were performed by flow cytometry. A total of 183 recipients were included in this current study. Among them, 84 (45.9%) were pre-transplant sensitized. Among the 84 sensitized patients, 28 patients received organs with UAGs as determined by Abs detected in undiluted serum samples. 13 of these 28 patients had positive HLA XM by flow cytometry. The one-year survival rate of the 56 sensitized patients who received organs without UAGs and the 28 sensitized patients who received organs with UAGs were equal, that is 89.3%. The one-year survival of the 99 non-sensitized patient is 92.9% (p = 0.685, comparing to that of sensitized patients). Among the 28 pts who received organs with UAGs determined in undiluted serum samples, the 1 year survival rate of patients with a positive XM (N = 13) is 84.6%, while the 1 year survival rate for the patients (N = 15) with a negative XM is 93.3% (p = 0.58). Based on our data, we conclude that the current approach of using a 1:16 titration to determine UAGs for the transplantation of sensitized heart patients is an acceptable practice. The long term survival beyond 1 year warrants further investigation.

P018 : IDENTIFICATION OF UNACCEPTABLE HLA ANTIGENS WHEN PRESENT IN TRANSPLANT PATIENT SERA AT AN UNDILUTED OR NEAT TITER BUT NOT PRESENT AT A LOW TITER (⩽1:16)

Characteristics identifying clinically deleterious HLA antibodies (Ab) may include specificity, mean fluorescence intensity (MFI) and C1q reactivity. These factors are also used when identifying unacceptable HLA antigens (Ags). However, if unacceptable HLA Ab specificities are present in undiluted sera but not present when sera are diluted 1:16 then these Ab specificities may be ignored when deciding on which unacceptable Ab specificities to report. This reduction in unacceptable Ags would allow for easier matching of highly sensitized patients (with high numbers of HLA Ab specificities) to an appropriate donor. Thirty-nine cI/cII high PRA sera were evaluated for the presence of HLA Abs and their Ag specificities at neat and at a 1:16 titer. The number of Ab specificities were compared at the two dilutions. One Lambda, Inc. reagents were used per the manufacturers instructions. The HLA cI/cII PRAs were 77 ± 25% and 84 ± 21% respectively for the 39 sera. The total number of HLA cI/cII Ags identified in undiluted sera were 46 ± 22 and 34 ± 22 respectively per patient. In addition, there were 7 ± 8 cI and 5 ± 4 cII falsely identified HLA Ags respectively per patient. When sera were titrated to a 1:16 dilution 71 ± 25% of cI and 51 ± 28% of cII unacceptable Ags were eliminated. The total number of cI Ags eliminated were 21 ± 10 and 15 ± 9 cII Ags. When false positive Ags were included in the evaluation the frequency for Ag elimination at a 1:16 dilution for cI became 85 ± 14% and 66 ± 24% for cII respectively. Fifty-four patients with donor specific antibody (DSA) titers of ⩽1:16 were identified. 81% (44/54) had both negative cytotoxic (AHG) and flow cytometric (FCXM) donor specific crossmatches whereas 10 patients (19%, 10/54) displayed a negative AHG but a positive FCXM. When transplanted, the FCXM negative patients had a two year graft survival of 91% vs. 60% for the FCXM positive patients ( p These data suggest that titration studies of high PRA, multiple Ab specificity presenting sera, can be used when identifying unacceptable Ags. Moreover, while the presence of DSA in low concentration (⩽1:16) may falsely suggest a positive virtual crossmatch it may not result in a real time positive crossmatch. HLA Ab specificities that are eliminated at a low titer (1:16) may allow for easier matching of highly sensitized patients to donors for transplant.

A 6-Year Experience of a Kidney Paired Donation Centralized Lab, Performing Crossmatches Resulting in 220 Transplants.

INTRODUCTION: Flow crossmatch (FXM) results with donor cells were compared to computer-based virtual crossmatch (VXM) results in patients stratified by their calculated PRA (cPRA) values to evaluate the accuracy of both crossmatch methods. METHODS: A total of 1949 VXM tests (with HLA-A, B, C, DR, and DQB) were performed. A single antigen bead assay was used for patients with 1-100% cPRA to create a list of unacceptable HLA antigens; VXM results were used to perform daily match runs. Selected donor/recipient pairs were tested by FXM with donor cells. Weekly match runs were performed to identify compatible pairs with negative VXM results. RESULTS: Between 2007 and 2012, there was an increase in the number of sensitized (1-79% cPRA) and highly sensitized (80-100% cPRA) patients (Figure 1). The accuracy of negative VXM ranged from 70.5% in 2007 to 74% in 2012 (Figure 2). False negative VXM results ranged from 26% in 2012 to 29.5% in 2007. When all negative VXM results were divided by cPRA, patients with 0% cPRA had no false negative results, those with 1-79% cPRA had 20% positive FXM, and those with 80-100% cPRA had 45% positive FXM. Analysis of the class I and II false negative VXM results revealed that the majority were falsely negative in both class I and II HLA (47%), rather than in class II alone (40%) or in class I alone (13%).Figure: No Caption available.Figure: No Caption available.CONCLUSION: Significant (˜ 25%) false negative VXM (especially for high PRA and Class II sensitized patients) when assessed by actual FXM demonstrates that data reported to centralized repositories by multiple transplant centers results in higher false negative VXM than can be achieved by single centers.

Center-Defined Unacceptable HLA Antigens Facilitate Transplants for Sensitized Patients in a Multi-Center Kidney Exchange Program

Multi-center kidney paired donation (KPD) is an exciting new transplant option that has not yet approached its full potential. One barrier to progress is accurate virtual crossmatching for KPD waitlists with many highly sensitized patients. Virtual crossmatch results from a large multi-center consortium, the National Kidney Registry (NKR), were analyzed to determine the effectiveness of flexible center-specific criteria for virtual crossmatching. Approximately two-thirds of the patients on the NKR waitlist are highly sensitized (>80% CPRA). These patients have antibodies against HLA-A (63%), HLA-B (66%), HLA-C (41%), HLA-DRB1 (60%), HLA-DRB3/4/5 (18-22%), HLA-DQB1 (54%) and HLA-DPB1 (26%). With donors typed for these loci before activation, 91% of virtual crossmatches accurately predicted an acceptable cell-based donor crossmatch. Failed virtual crossmatches were attributed to equivocal virtual crossmatches (46%), changes in HLA antibodies (21%), antibodies against HLA-DQA (6%), transcription errors (6%), suspected non-HLA antibodies (5%), allele-specific antibodies (1%) and unknown causes (15%). Some failed crossmatches could be prevented by modifiable factors such as more frequent assessment of HLA antibodies, DQA1 typing of donors and auditing data entry. Importantly, when transplant centers have flexibility to define crossmatch criteria, it is currently feasible to use virtual crossmatching for highly sensitized patients to reliably predict acceptable cell-based crossmatches.

Elimination of Unacceptable HLA Antigens When Present in Transplant Patient Sera at an Undiluted or Neat Titer But Not Present at a Low Titer (≤ 1:16).

HLA antibodies (Ab) are routinely identified in transplant recipient sera. Characteristics determining clinically deleterious Abs include specificity, MFI and C1q. These factors are also used when identifying unacceptable HLA antigens (Ags). However, if unacceptable HLA Ab specificities are present in undiluted sera but not present when the sera is diluted 1:16 then these Ab specificities can possibly be ignored when deciding on which unacceptable Ab specificities to report. This reduction in unacceptable antigens would allow for an easier matching of highly sensitized patients to an appropriate donor for transplant. Thirty-nine cI/cII high PRA sera were evaluated for the presence of HLA Abs and their antigen specificities at neat and at a 1:16 titer. The number of Ab specificities were compared at the two dilutions. One Lambda, Inc. reagents were used per the manufacturers instructions. The HLA cI/cII PRAs were 77±25% and 84±21% respectively for the 39 sera. The total number of HLA cI/cII Ags identified in undiluted sera were 46±22 and 34±22 respectively per patient. In addition, there were 7±8 cI and 5±4 cII falsely identified HLA Ags respectively per patient. When sera were titrated to a 1:16 dilution 71±25% of cI and 51±28% of cII unacceptable Ags were absent. The total number of cI Ags eliminated were 21±10 and cII Ags were 15±9. When false positive Ags were included in the evaluation the frequency for Ag elimination at a 1:16 dilution for cI became 85±14% and 66±24% for cII respectively. Fifty-four patients with donor specific antibody (DSA) titers of ≤ 1:16 were identified. 81% (44/54) had both negative cytotoxic (AHG) and flow cytometric (FCXM) donor specific crossmatches whereas 10 patients (19%, 10/54) displayed a negative AHG but a positive FCXM. When transplanted, the FCXM negative patients had a two year graft survival of 91% vs. 60% for the FCXM positive patients (p < 0.01). These data suggest that titration studies of high PRA, multiple Ab specificity presenting sera can be used when identifying unacceptable Ags. Moreover, the presence of DSA in low concentration (≤ 1:16) while falsely suggesting a positive virtual crossmatch may not result in a real time positive crossmatch. HLA Ab specificities that are eliminated at a low titer (1:16) may allow for easier matching of highly sensitized patients to donors for transplant.

Current Status of Heart Transplantation and Left Ventricular Assist Device: Major Changes in the Last Decade

Heart transplantation is the last treatment option in refractory end stage heart failure, which can prolong survival. The number of heart transplantations has increased and the survival rate has improved during the last few decades which was contributed by advanced understanding of immunologic mechanism of rejection, pharmaceutical development and clinical management of donors and recipients. However, only a fraction of patients can be offered to transplantation due to shortage of donor heart and many patients suffer high mortality while waiting. Meanwhile, technical advancement of mechanical assist device in recent years enabled long term implantable left ventricular assist devices (LVAD) to bridge the patients with high mortality in the waiting list to transplantation and to assist as a long term destination therapy for patients who are not eligible for transplantation. Development of solid phase assay increased the sensitivity and the specificity of detection of anti-human leukocyte antigen (HLA) antibodies in the recipient. It enabled identifying unacceptable HLA antigens, acquire calculated Panel Reactive Antibodies and perform virtual cross match that can enhance the efficacy of donor allocation system to decrease the waiting time, obviate prospective cross match to decrease ischemic time and to assess the risk of rejection in presensitized patients. Antibody mediated rejection is a challenging entity in diagnosis and management. However, standardized classification of histology and immunology of endomyocardial biopsies was made recently and immunotherapy is moving toward targeted therapies directed at antibody production and function. This review focuses on those major changes in the heart transplantation field in the last decade.

Calculated PRA: Initial Results Show Benefits for Sensitized Patients and a Reduction in Positive Crossmatches

The calculated panel reactive antibody (CPRA), which is based upon unacceptable HLA antigens listed on the waitlist form for renal transplant candidates, replaced PRA as the measure of sensitization among US renal transplant candidates on October 1, 2009. An analysis of the impact of this change 6 months after its implementation shows an 83% reduction in the number of kidney offers declined nationwide because of a positive crossmatch. The increasing acceptance and utilization of unacceptable HLA antigens to avoid offers of predictably crossmatch-positive donor kidneys has increased the efficiency of kidney allocation, resulting in a significant increase in the percentage of transplants to broadly sensitized (80+% PRA/CPRA) patients from 7.3% during the period 07/01/2001-6/30/2002 to 15.8% of transplants between 10/1/09-3/31/10. The transplant rates per 1000 active patient-years on the waitlist also increased significantly for broadly sensitized patients after October 1, 2009. These preliminary results suggest that 'virtual' positive crossmatch prediction based on contemporary tools for identifying antibodies directed against HLA antigens is effective, increases allocation efficiency and improves access to transplants for sensitized patients awaiting kidney transplantation.

Role of Flow Cytometry to Define Unacceptable HLA Antigens in Lung Transplant Recipients with HLA-Specific Antibodies

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. 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. 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. 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.