Unacceptable Antigens Research Articles

Auto- and allo-epitopes in DQ alloreactive antibodies.

Significant interest is now focused on deciphering human leukocyte antigen (HLA) epitopes and the utilization of this new knowledge to improve donor-recipient matching in transplantation. A recently introduced concept is the appearance of antibodies against what may be considered as self-epitopes, including the introduction of the 'nonself-self paradigm'. Common practice in analyzing HLA-DQ antibodies have been to separate between antibodies against the α chain and antibodies against the β chain of the molecule. This is despite the fact that the two chains have to intertwine together to be expressed stably on the cell surface. We have previously provided evidence that this practice is false. We further provide evidence to refute the use of 'self-epitopes' as an immunologically feasible terminology by delineating the historic events leading to today's misconceptions. The evidence we present supports the need for a change in current practices. HLA-DQ antigens and antibodies should be viewed as combined DQα/β complexes. This will have impact to assigning cPRA value, assigning acceptable and unacceptable antigens, and pave the way to a better understanding of true HLA epitopes as we strive to improve donor--recipient compatibility and minimize generation of de-novo HLA antibodies.

Many de novo donor-specific antibodies recognize β2 -microglobulin-free, but not intact HLA heterodimers.

Solid‐phase single antigen bead (SAB) assays are standard of care for detection and identification of donor‐specific antibody (DSA) in patients who receive solid organ transplantation (SOT). While several studies have documented the reproducibility and sensitivity of SAB testing for DSA, there are little data available concerning its specificity. This study describes the identification of antibodies to β2‐microglobulin‐free human leukocyte antigen (β2‐m‐fHLA) heavy chains on SAB arrays and provides a reassessment of the clinical relevance of DSA testing by this platform. Post‐transplant sera from 55 patients who were positive for de novo donor‐specific antibodies on a SAB solid‐phase immunoassay were tested under denaturing conditions in order to identify antibodies reactive with β2‐m‐fHLA or native HLA (nHLA). Antibodies to β2‐m‐fHLA were present in nearly half of patients being monitored in the post‐transplant period. The frequency of antibodies to β2‐m‐fHLA was similar among DSA and HLA antigens that were irrelevant to the transplant (non‐DSA). Among the seven patients with clinical or pathologic antibody‐mediated rejection (AMR), none had antibodies to β2‐m‐fHLA exclusively; thus, the clinical relevance of β2‐m‐fHLA is unclear. Our data suggests that SAB testing produces false positive reactions due to the presence of β2‐m‐fHLA and these can lead to inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post‐transplant period.

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.

CPRA for allocation of kidneys in the US: More candidates ≥98% CPRA, lower positive crossmatch rates and improved transplant rates for sensitized patients

In 2009 calculated panel reactive antibody (CPRA) replaced PRA as the metric for HLA sensitization in the US kidney allocation system. During the next four years, registrants with at least one unacceptable antigen increased (34–40%) and registrants with ≥98% PRA/CPRA increased from 7% to 9% of the waitlist. These changes were accompanied by a reduction in kidney offers refused for positive crossmatch: 14,137 (1.7%) in 2009 and 3,310 in 2013 (0.4%). Registrants with ≥98% PRA/CPRA had highest rates of refusal but also showed substantial improvement (20% in 2009 vs 8% in 2013). For registrants with ≥98% PRA/CPRA, 45% of accepted offers in 2009 were not transplanted into the intended recipient compared to 11% in 2013. Transplant rates remained low for these patients (∼50/1000 active patient-years), but rates improved for patients with 80–97% PRA/CPRA (223/1000 active patient-years in 2009 vs 354/1000 in 2013). In 2013, 40% regraft candidates had CPRA ≥98% compared to 4% of primary graft candidates. More females than males were ≥98% CPRA (14% vs 7%) and more females had CPRA above 0 (50% vs 28%). In the CPRA era, listing of unacceptable antigens increased, positive crossmatches were diminished and transplant rates for sensitized patients improved.

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.

Single antigen flow beads for identification of human leukocyte antigen antibody specificities in hypersensitized patients with chronic renal failure

Aims of this studyAims of this study were to identify class I and class II antibodies in highly sensitized patients by flow cytometry single antigen bead (FC-SAB) assay and to evaluate according to donor HLA type in order to increase their kidney transplantation chance.Material and methodsWe analyzed 60 hypersensitive patients of 351 individuals, who applied to our laboratory for PRA test in November 2013-December 2014. Flow cytometric PRA screening and single antigen bead commercial kits were used for these analyses.ResultsIn our study group, 19 (31.7%) of these patients were male while 41 (68.3%) patients were female. The most common acceptable antigens were A*02 (10.11%), HLA-A*23 (10.11%), HLA-B*38 (8.79%) and HLA-DRB1*03 (7.83%) in hypersensitive patients. The highest antibody reactivity on SAB was observed against HLA-A*25, HLA-B*45, HLA-DRB1*04 and HLA-DRB1*08 antigens.ConclusionsThe determination of these acceptable and unacceptable antigens may increase their transplantation chance. Pre-transplant HLA antibody identifications provide prognostic information with respect to the determination of patients who are at increased risk of graft loss.

Cost-Effectiveness of Pediatric Heart Transplantation Across a Positive Crossmatch for High Waitlist Urgency Candidates

Allosensitized children listed with a requirement for a negative prospective crossmatch have high mortality. Previously, we found that listing with the intent to accept the first suitable organ offer, regardless of the possibility of a positive crossmatch (TAKE strategy), results in a survival advantage from the time of listing compared to awaiting transplantation across a negative crossmatch (WAIT). The cost-effectiveness of these strategies is unknown. We used Markov modeling to compare cost-effectiveness between these waitlist strategies for allosensitized children listed urgently for heart transplantation. We used registry data to estimate costs and waitlist/posttransplant outcomes. We assumed patients remained in hospital after listing, no positive crossmatches for WAIT, and a base-case probability of a positive crossmatch of 47% for TAKE. Accepting the first suitable organ offer cost less ($405 904 vs. $534 035) and gained more quality-adjusted life years (3.71 vs. 2.79). In sensitivity analyses, including substitution of waitlist data from children with unacceptable antigens specified during listing, TAKE remained cost-saving or cost-effective. Our findings suggest acceptance of the first suitable organ offer for urgently listed allosensitized pediatric heart transplant candidates is cost-effective and transplantation should not be denied because of allosensitization status alone.

Optimizing transplantation of sensitized heart candidates using 4 antibody detection assays to prioritize the assignment of unacceptable antigens

The virtual crossmatch relies on the assignment of unacceptable antigens (UAs) to identify compatible donors. The purpose of our study was to identify an algorithm for assignment of UAs such that a negative complement-dependent cytotoxicity (CDC) crossmatch and concomitant negative or weakly positive flow cytometric crossmatch (FXM) are obtained. We used 4 antibody methods: (1) Luminex single antigen (LSA), (2) LSA with a 1:8 serum dilution, (3) C1q LSA, and (4) CDC panel. The UAs were prioritized in the following order: (1) all C1q+/CDC+, (2) LSA 1:8 >7,500 median fluorescence intensity, and (3) LSA >10,000 median fluorescence intensity. Of 295 heart transplants that were performed at our center, 69 (23%) recipients had detectable human leukocyte antigen specific antibody at the time of transplant. All donor specific antibodies (DSAs) were avoided for 44 of 69 (64%) (DSA-). There were 25 recipients who had DSA at the time of transplant: 12 (48%) had negative FXM (DSA+/FXM-), and 13 (52%) had positive T-cell and/or B-cell FXM (DSA+/FXM+). Lower freedom from antibody-mediated rejection was observed for the DSA+/FXM+ group compared with the DSA- group (p < 0.0001). DSA remained detectable after transplant in the sera of 14 recipients, and de novo DSA was detected in 32 recipients. Freedom from antibody-mediated rejection was comparable for both groups (p = 0.53) but was lower than the DSA- group (p < 0.0001). Survival was comparable for all groups at 1,200 days post-transplant. Strategic prioritization of UA assignment has allowed transplantation of highly sensitized patients across the DSA barrier with survival rates comparable to DSA- heart transplant recipients.

Determination of unacceptable HLA antigen mismatches in kidney transplant recipients: recommendations of the German Society for Immunogenetics.

One of the major tasks of histocompatibility and immunogenetics laboratories is the pretransplant determination of unacceptable antigen mismatches (UAM) in kidney transplant recipients. In this procedure, human leucocyte antigen (HLA) specificities are defined against which the patient has circulating alloantibodies that are expected to harm the transplanted organ. Using the information on UAM and the potential donor's complete HLA typing, prediction of the crossmatch result, the so called 'virtual crossmatch', is possible. Currently, the laboratories are using different algorithms for the determination of UAM, and depending on the algorithm, more or fewer organ offers are excluded for patients with a similar antibody profile. In order to bring homogeneity into the allocation of organs to immunized patients in Germany, the German Society for Immunogenetics established, on the basis of current knowledge, recommendations for the determination of UAM. The UAM recommendations, which are thought to serve as a common tool for responsible physicians at different transplant centers, contain technical issues that need to be considered and are individualized for sensitized patients with a high or intermediate risk of antibody-mediated rejection. The present review contains these recommendations and puts them into perspective to current international practice.

Why do some 100% CPRA renal transplant candidates get more offers with the new KAS than others?

Aim In the 5 months after implementation of the new kidney allocation system (KAS), import kidney offers to 100% cPRA renal transplant candidates (RTC) have increased due to prioritization with an additional 212 points. We noted that while some RTC were receiving multiple provisional offers, other patients were not receiving any. We hypothesized that the patients receiving fewer offers had more unacceptable antigens (UA) entered in UNet, had a less common blood type, or had UA against broad specificities. Methods Active RTC with 100% cPRA from a single center were examined for: total numbers of class I and class II UAs; high frequency broad UAs such as Bw4, Bw6, DR52, etc.; wait time on 12/4/14; expected post-transplant survival (EPTS) score; and ABO blood type. DQA and DPB UAs were also examined, although they can’t be entered in UNet. RTC were stratified by number of offers into 3 groups (0, 1–2, and ⩾ 4 offers), with differences analyzed using Students t-test. Three RTC transplanted within 1 month of KAS were omitted. Results Comparisons are shown in Table 1. The group with 0 offers had a significantly greater number of class I UAs compared to the other groups. For class II UAs, the 0 and 1–2 groups had a significantly greater number than the ⩾ 4 group (p = 0.002). Closer analysis of class II UAs showed that the ⩾ 4 group had a lower incidence of DR52 UA (17%) vs the other groups (50% and 63%). DR51 UA had an incidence of 50% in the 2 groups with the fewest offers, compared to 33% in the ⩾ 4 group. DR53, Bw4, Bw6, Cw, DQ, DPB, mean EPTS score, wait time, and ABO were not significantly different between groups. Conclusion Although this is a single center study with small numbers, these findings may be reproduced with national data. RTC with many UAs may have an extremely narrow window of acceptable mismatches (MM). Donors with 0 MM at one or more loci or with unusual HLA phenotypes may be their only source of offers. Additional strategies may be needed, including sufficient additional points to achieve allocation when there are 0MM at the HLA-A or -B loci. Download : Download full-size image

The new kidney allocation system-who are we penalizing?

The new kidney allocation system implemented at the end of 2014 attributes additional allocation points to sensitized patients using a sliding cPRA scale. In addition patients with > 98% cPRA receive regional/national allocation priority due to their high level of sensitization. Unfortunately,the new allocation system does not allow for the input of HLA-DQA and HLA-DP unacceptable antigens for the computation of cPRA. To assess the number of patients affected by the lack of programming for HLA-DQA and -DP, we reviewed the antibody data of our adult kidney waitlist population composed of 485 patients. We found that 7.6% of our patients had HLA-DP and/or HLA-DQA antibodies with MFI values > 3000 for which they were not receiving additional allocation points due to our inability to enter HLA-DQA and -DP specificities as unacceptable antigens. The vast majority of the affected patients (86.5%) had HLA-DP antibodies and cPRA of 98%. In fact 75.7% of patients with HLA-DQA or HLA-DP antibodies had cPRA of 98% while 24.3% had cPRA > 98% and thus were eligible for regional/national priority. Our gathered data strongly advocates the implementation of new allocation software allowing for the listing of HLA-DQA and HLA-DP unacceptable antigens and for computation of these unacceptable antigens in the cPRA. Per OPTN data as of 4/22/15 there were 123,193 people waiting for a kidney transplant in the US. Using our center specific data on a national scale, it means that 9300 people are not receiving allocation points for unacceptable antigens against HLA-DP and/or -DQA. Our data also supports the implementation of mandatory typing of donors for HLA-DP and DQA since 24.3% of patients with HLA-DQA and -DP antibodies have cPRA > 98%. Mandatory typing of donors for HLA-DP and -DQA would allow for faster organ allocation and unnecessary shipment of organ for regional/national share to patient having unacceptable HLA-DQA or -DP antibodies.

Assessing the impact of the new kidney allocation system on rates of transplantation for highly sensitized recipients – Center specific data

Background The new OPTN/UNOS kidney allocation system (KAS) is designed to expand donor access for highly-HLA sensitized patients (HS). Our approach to transplanting the HS patients (PTS) awaiting deceased donor (DD) transplants (TXP) is to assign unacceptable antigens (UA) based on 4 antibody testing methods and to desensitize PTS using high dose IVIG and Rituximab. In the past, we would assign priority based on wait time and likelihood of receiving DD offers. Aim The aim of our study was to assess the impact of the new KAS on TXP rates for PTS with varying levels of sensitization compared to non-sensitized PTS. We examined PTS who are HS (99/100CPRA), moderately sensitized (80–98 CPRA) and less sensitized ( 20CPRA). Methods UAs are assigned based on 4 methods luminex single antigen with and without dilution, C1Q assay and CDC. Final crossmatches are performed by Flow and CDC. Patient’s antibody history, CPRA and crossmatch results were tabulated for this study. Rates of deceased donor TXP for a period of 144 days prior to and after December 7, 2014 were analyzed. Results The rate of transplantation for PTS in the 99–100% CPRA group was the same in both time periods. The number of deceased donor TXP in the 80–98% and 20–79% CPRA groups was reduced by 50%. However the number of TXP for PTS in 0–19% CPRA was increased by 50%. Conclusions While the rate of deceased donor TXP for PTS in 99–100% CPRA at our center remained the same at ∼ 12.7%, it increased from 2.5% to 17.7% for the national average. The new KAS appears to reduce the chance of transplantation for moderately sensitized recipients and increases the number of TXP for non-sensitized recipients at our center. The KAS appears to have little impact on transplanting the highly sensitized PTS at a center with progressive assignment of UAs and a desensitization program. Download : Download full-size image

Evaluation of discrepancies between Luminex single antigen bead assays

Currently there are two manufacturers offering HLA antibody testing using the Luminex platform. Our laboratory incorporates both methods in our testing protocol. We have documented discrepancies and use them to help us identify samples that need follow-up verification of unacceptable antigens. More discrepancies are found with Class I than with Class II and some appear to be major differences. The purpose of this study was to examine the discrepancies with Class I single antigen testing between the two manufacturers with respect to correlation with Flow crossmatch results and MFI values. For this study discrepant samples from a single run of Class I single antigen beads were chosen. An antibody of interest was chosen for each sample and surrogate cells were found which did not have HLA detected by other Class I antibodies reported in the same sample. Flow crossmatch was performed using a BD FACsCaliber instrument on a 1024 scale. All discrepancies in this study resulted from an antibody being called by Method A that was not detected using Method B. Surrogate crossmatches were done to determine if there was a sensitivity problem with Method B or a specificity problem with Method A. Twenty-seven crossmatches were examined which included eleven different specificities. Fourteen samples were T cell positive and thirteen were T cell negative. Of the fourteen that were T cell positive, six were B cell negative, which could indicate non-HLA reactivity. Therefore, it is possible that 19 of the 27 specificities detected by Method A may not be clinically relevant. The discrepancies between methods is of concern and emphasizes the necessity of including manufacturers and MFI cutoffs when publishing Luminex single antigen bead results. Most HLA laboratories use single antigen results for determining unacceptable antigens and virtual crossmatches. More specificities were called with Method A using our current cutoff, but at least half of these did not result in a positive Flow crossmatch. Re-evaluation of the cutoff suggests that the cutoff may be different for certain loci or certain specificities. The use of both methods may have value in fine-tuning the unacceptable antigens and illuminating those specificities that should be confirmed.

The new OPTN Kidney Allocation System: An early look at the transplant trend in the highly sensitized patient cohort

Aim The new OPTN Kidney Allocation System (KAS) is expected to prioritize highly sensitized candidates nationwide. Many of these candidates are likely to have antibodies to DP and/or DQA antigens, which are not accounted for in the assessment of cPRA and unacceptable antigens listed in UNOS. This can compromise the feasibility of performing reliable virtual crossmatches (XMs). Herein, we evaluated the impact of new KAS thus far, on kidney allocation for highly sensitized candidates of cPRA ⩾ 98%. Methods Data was collected from the 8 renal transplant centers in Michigan for recipients with cPRA ⩾ 98%, between Dec 4, 2014 and Apr 6, 2015 and the same 4-month period of the previous four years. In this cohort, we evaluated: (1) number of kidney offers; (2) frequency of DP/DQA antibodies; (3) “unexpected” positive XMs; (4) transplant rates; (5) number of out-of-state kidney offers; and (6) kidney discard rate. Results The number of kidney offers to recipients with cPRA ⩾ 98% revealed a 160% increase, from 5 pre-KAS to 13 post-KAS offers/month. Antibodies to DP and/or DQA were present in 58% of these recipients (30/52), and “unexpected” B-cell positive XMs due to such antibodies were observed in 30% of the XMs (9/30). Overall, the transplant rate for this group increased significantly to 42% (22 transplanted/52 offers) in comparison to a total of 13% (11/82) from the same time period in the past 4 years. Approximately, 1/3 of transplanted recipients from the new-KAS cohort had DP/DQA antibodies. In addition, since the new KAS, out-of-state kidney offers increased significantly by 11-fold to 33 in the 4-month period compared to that of the previous 4 years. Lastly, kidney discard rate slightly elevated to 22% (10 discards/46 kidneys) compared to an average of 17% (4/23), which could in part be attributed to the “unexpected” positive XMs. Conclusions To date, our data suggested that despite the unaccounted DP/DQA antibodies and “unexpected” positive XMs, the new KAS has enhanced organ allocation for the highly sensitized candidates, signified by the increased transplant rate. At this pace, we project 66 of these recipients in Michigan will be transplanted by year’s end. The increase in kidney discard rate especially due to positive XMs warrants further attention.

Defining the non-sensitized patient: a comparison of HLA antibody screening and identification methods

Aim Luminex Single Antigen beads (SAB) are widely used to identify HLA specificities and assign avoid antigens for kidney transplantation. However in our centers, “non-sensitized” waitlist patients are monitored using less sensitive screening kits. To confirm the lack of HLA sensitization, we evaluated sera by SAB. In this study, we compared several antibody screening and identification kits to uncover test discrepancies and investigate possible false reactions. Methods During initial evaluation and listing, patients were defined as “non-sensitized” when the Class I/II FlowPRA screening beads (One Lambda) were negative. Presumed non-sensitized waitlist patients are screened monthly by the Lifecodes Lifescreen kit (Immunocor) and screened yearly by FlowPRA screening beads. To ensure that no HLA antibodies were missed by this protocol, 116 patient samples were tested by One Lambda Class I/II Labscreen Single Antigen beads (1000 MFI cutoff). When discrepancies between the three assays were identified, sera were tested by the Immunocor ID assay and/or One Lambda Labscreen PRA beads. Additional analyses by surrogate crossmatches are ongoing. Results Of the 116 samples tested from non-sensitized patients, 22% were positive by the Labscreen Class I SAB and 33% were positive by the Labscreen Class II SAB but negative by the screening kits. The positive specificities spanned all HLA loci, but there were some beads that were positive more than expected. Most notably, the DRB1∗04:04 bead was positive in 10% of cases (12/116) with a range of 1080–2524 MFI, and in three cases the patient’s HLA type was DR4. Other specificities observed three or more times included: A80, B45, B76, Cw15, DR18, DP1, DP11, bead DQA1∗01:04/DQB1∗06:04. In four cases, patients presented with new UNet unacceptable antigens (⩾3000 MFI), and in three cases this led to a positive cPRA (4–17%). Conclusions Unexpected positive specificities were detected by the Labscreen SAB assay in otherwise non-sensitized kidney waitlist patients. Our data are consistent with other studies that compared multiple test assays to interrogate false reactions. Unexpected SAB reactions should be investigated with additional screening assays and/or surrogate crossmatches. In some cases, common false positive beads can be ignored during test interpretation.

HLA Antibody testing- it’s not a one stop assay

HLA laboratories rely on single antigen (SA) assays to determine unacceptable antigens and perform virtual crossmatches (vXM) that can immunologically guide clinicians in the identification of an appropriate donor. Although solid phase antibody detection assays are sensitive, specific and robust-instances arise when further testing is needed to verify results or resolve inconsistencies between these tests. Patient S.T. is a 50 year old male diagnosed with heart failure. Routine work-up in our laboratory is to evaluate % FlowPRA and compare those results to solid phase assays on a Luminex platform which measures the breadth and strength of antibodies in a patient’s sera. Patient S.T. had two samples tested three months apart with a class I FlowPRA of 58% and 62%, respectively. However, The Luminex SA results consistently demonstrated only 5 class I (all B15) antibodies in the positive MFI range leading to a cPRA of about 15%. Moreover, the internal negative control values were > 2500 MFI leading us to believe that there may be additional specificities masked by the high background. How should we deal with these contradictory test results? Can we rely on a vXM to accept a heart for this patient? To reconcile these seemingly contradictory results and to determine if the antibodies detected by Luminex assays were relevant, surrogate FCXM were performed. Surrogate Donor 1 (B15 positive) was FCXM positive. Surrogate donor 2 (B15 negative) was FCXM negative indicating that non-specific binding/false-positivity due to high background should not affect FCXM interpretation, Importantly, testing using phenotype beads were not affected by high background and further confirmed the presence of B15 antibodies. Due to the presence of HLA antibodies and the high background exhibited by this patient, a prospective FCXM must be performed prior to accepting an organ. In conclusion, this is an example in which different tests provide contradictory (or non-consistent) results - and our approach to resolve these ambiguities to support successful organ transplantation.

Luminex-based Immunoassay for Organ Transplantation

Development of luminex-based solid phase assays enables advanced measurement of HLA antibody with sensitivity, specificity, and increasing knowledge of unacceptable antigens. In this review, we described the principle of the luminex-based assay and its current applications for organ transplantation including C1q assay, calculated panel reactive antibody, and virtual crossmatching. We also discussed the technical aspects and limitations for clinical utilization. The variables related to measurement of HLA antibody specificities and their clinical relevance remain unclear, therefore the interpretation of results requires comprehensive knowledge and clinical information in critical cases.

CPRA Increases With DQA, DPA, and DPB Unacceptable Antigens in the Canadian cPRA Calculator.

A calculated panel reactive antibody (cPRA) estimates the percentage of donors with unacceptable antigens (UA) for a recipient. cPRA may be underestimated in transplant candidates with UA to DQA, DPA, and DPB if these are not included in the calculation program. To serve the National Canadian Transplant Programs, a cPRA calculator was developed with complete molecular typing for all donors at HLA‐A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1, all resolved to serologic equivalents. The prevalence of UA at DQA, DPA and DPB was evaluated in a sensitized regional population. The impact of adding these additional UA to cPRA was calculated alone and in combination, and compared to the baseline cPRA for UA at A, B, C, DR, DR51/52/53, and DQ. Of 740 sensitized transplant candidates, 18% of total and 32% with cPRA≥95% had DQA UA. Twenty‐seven percent of total and 54% with cPRA≥95% had DPB UA. Of 280/740 subjects with these UA, 36/280 (13%) had cPRA increase of >20% when they were included, 7% increased cPRA to ≥80% and 6% to ≥95%. Inclusion of DQA, DPA, and DPB UA in Canadian cPRA calculations improves the accuracy of cPRA where these are relevant in allocation.

(798) - Identification of Clinically Relevant Unacceptable Antigens By Use of Titration Studies of Heart Transplant Patient Sera

Patients listed for transplantation (Tx) have a calculated panel reactive antibody (CPRA) that is derived from the HLA antibody (Ab) specificities present in their sera. Unacceptable antigen (UAg) identification is done by testing undiluted sera. However, titration studies have shown some UAgs to be eliminated at a 1:16 titer suggesting a potentially irrelevant specificity.

LBP06: HLA-C ANTIBODY: HOW STRONG IS UNACCEPTABLE? DEFINING HLA-C ANTIBODY CUT-OFF AT ONE TRANSPLANT CENTER

The addition of HLA-C to UNOS cPRA calculation was the result of increased recognition of the importance of HLA-C antibody and the fact that enough data was available on the frequency of HLA-C types in donors. The unacceptable cut-off for HLA-A/B/DR/DQ antibodies, which varies drastically throughout the HLA field, was determined based on the likelihood of producing a positive crossmatch. Will the same arbitrary cut-off apply to HLA-C? HLA-C has unique antigen expression level on the cell surface and limited antigenic polymorphism, which can create artificial over representation on Luminex Single antigen beads. This is an ongoing case study from one transplant center aiming to answer these questions. Nine sera with HLA-C antibodies were identified; five of them only had HLA-C antibodies. The antibody profile and strength were characterized using Luminex single antigen bead assay (One Lambda Inc, Canoga Park, CA). Surrogate donor cells with corresponding DSA were collected and crossmatched using CDC, AHG-CDC and Flow methods. All HLA-C DSA(s) are >4000 MFI, which is the cut-off used in our center as unacceptable antigen determination and likely to produce positive CDC and/or AHG-CDC crossmatch. All CDC, AHG-CDC crossmatches were negative. Among the 4 sera with multiple loci DSA, 3 were T and B cell Flow positive and 1 was B cell positive only. Among the 5 HLA-C DSA only sera, 3 were T and B cell Flow positive and 2 were T and B cell Flow negative. Anti-Cw4 demonstrated positive flow T and B cell crossmatches with MFI’s over 4K range (5–8 K tested). Anti-Cw1, often observed as only HLA-C antibody sera together with Cw1/C15/C12, was completely negative for all T and B cell CDC, AHG-CDC and Flow crossmatches. If HLA-C unacceptable antigen cut-off is set at 4000 MFI, all of the above ‘donors’ would have been eliminated. At a center where transplants are routinely performed over positive DSA/Flow crossmatch, all of these cases would be transplantable, and furthermore, two of these cases would not require any immune modulation prior to transplant. An anti-Cw7 calculates as 49% cPRA and will cause a high percentage of donor exclusion. Is the Cw1/C15/C12 valid? Ongoing data collection is in progress for better defining HLA-C antibody and unacceptable cut-off for virtual donor exclusion.