Positive Virtual Crossmatch Research Articles

P037 : LACK OF C1Q REACTIVITY IN POST-LVAD PATIENT SERA

End stage heart disease can be treated with implantation of a mechanical circulatory support device to assist the left ventricle (LVAD) as a bridge to heart transplantation. During the LVAD operation patients may be exposed to large volumes of blood and blood products resulting in production of anti-HLA antibodies (Abs). This higher degree of HLA antigen exposure/sensitization may result in positive virtual crossmatches, eliminating many potential donor-recipient pairings for transplantation. However, when LVAD patients are transplanted they do not have a different rejection frequency or poorer graft outcome than non-LVAD or non-sensitized heart transplant recipients. We hypothesized that while these LVAD treated patients present with anti-HLA Abs these Abs may not be C1q reactive and therefore may not be functionally deleterious. We evaluated 90 patients (61 male and 29 female with a mean age of 54 years). Seventy-three patient sera were evaluated pre-LVAD and 17 patient sera post-LVAD for the presence of cI and cII HLA Abs and C1q reactivity using One Lambda, Inc. reagents and following manufacturer’s instructions. The cI and cII PRAs pre and post LVAD were divided into three groups. The cI PRAs of 0–5% were seen in 57% of patients pre-LVAD vs. 59% of patients post-LVAD. PRAs of >5–50% were seen in 23% of patients pre-LVAD vs. 29% of patients post-LVAD. Finally, PRAs of >50% were seen in 20% of patients pre-LVAD vs. 12% of patients post-LVAD. Class II PRAs of 0–5% were seen in 83% of patients pre-LVAD vs. 59% of patients post-LVAD. Class II PRAs of >5–50% were seen in 7% of patients pre-LVAD vs. 29% of patients post-LVAD ( p ⩽ 0.05). Finally, PRAs of >50% were seen in 10% of patients pre-LVAD vs. 12% of patients post-LVAD. Pre-LVAD C1q reactivity was seen in 38% of patients with cI HLA Abs and 45% of patients with cII HLA Abs. However, in contradistinction to the pre-LVAD C1q positive reactivity only 8% of both cI and/or cII post-LVAD sera were C1q positive (8% vs. 38%, p < 0.01; 8% vs. 45%, p < 0.01). These data suggest that while HLA antibodies are present in post-LVAD patient sera 92% of these antibodies tested as C1q non-reactive. This may explain why these identifiable HLA antibodies do not adversely impact LVAD treated heart transplant patients.

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.

Lack of C1q Reactivity in Post-LVAD Patient Sera.

End stage heart disease can be treated with implantation of a mechanical circulatory support device, often to assist the left ventricle (LVAD) as a bridge to heart transplantation. During the LVAD operation patients may be exposed to large volumes of blood and blood products resulting in production of anti-HLA antibodies (Abs). This higher degree of HLA sensitization results in positive virtual crossmatches, eliminating many potential donors. However, when LVAD patients are transplanted they do not have a different rejection severity or frequency or poorer graft outcome than non-LVAD or non-sensitized heart transplant recipients. We hypothesized that while these LVAD treated patients display anti-HLA Abs these Abs may not be C1q reactive and therefore may not be functionally deleterious. We evaluated 90 patients (61 male, 29 female, mean age of 54 years). Seventy-three patient sera were evaluated pre-LVAD and 17 patient sera post-LVAD for the presence of cI and cII HLA Abs and C1q reactivity using One Lambda, Inc. reagents and following manufacturers instructions. The cI and cII PRAs pre and post LVAD were divided into three groups. For cI PRAs of 0-5% were seen in 57% of patients pre-LVAD vs. 59% of patients post-LVAD. PRAs of > 5-50% were seen in 23% of patients pre-LVAD vs. 29% of patients post-LVAD. Finally, PRAs of > 50% were seen in 20% of patients pre-LVAD vs. 12% of patients post-LVAD. Class II PRAs of 0-5% were seen in 83% of patients pre-LVAD vs. 59% of patients post-LVAD. Class II PRAs of > 5-50% were seen in 7% of patients pre-LVAD vs. 29% of patients post-LVAD (p≤0.05). Finally, PRAs of >50% were seen in 10% of patients pre-LVAD vs. 12% of patients post-LVAD. Pre-LVAD C1q reactivity was seen in 38% of patients with cI HLA Abs and 45% of patients with cII HLA Abs. However, in contradistinction to the pre-LVAD C1q positive reactivity only 8% of both cI and/or cII post-LVAD sera were C1q positive (8% vs. 38%, p<0.01; 8% vs. 45%, p<0.01) These data suggest that while HLA antibodies are present in post-LVAD patient sera 92% of these antibodies tested as C1q non-reactive. This may explain why these identifiable HLA antibodies do not adversely impact LVAD treated heart transplant patients.

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.

(844) - Impact of Pre-Sensitization and Positive Virtual Cross-Match on Outcomes in Pediatric Heart Transplantation

(844) - Impact of Pre-Sensitization and Positive Virtual Cross-Match on Outcomes in Pediatric Heart Transplantation

LVAD Implant as a Bridge to Heart Transplantation Is Associated with Allosensitization as Measured by Single Antigen Bead Assay

Left ventricular assist devices (LVAD) as a bridge (BTT) to heart transplantation (HTX) may be limited by the formation of anti-human leukocyte antigen antibodies. Whether sensitization occurs with continuous axial flow LVAD implant as assessed by single antigen bead (SAB) assay is unknown. Cytotoxic panel-reactive antibody (PRA) and SAB assays were analyzed in HTX recipients undergoing LVAD implant as a BTT. Sensitization was defined as peak anti-human leukocyte antigen antibody values of more than 2000 mean fluorescence intensity because these values have been found to correlate with flow cytometric crossmatch results. LVADs were implanted as BTT in 30 patients. There were 7% (2 of 30) of patients before LVAD implant and no patients after LVAD implant with PRA more than 10%. However, 20% (6 of 30) of patients before LVAD and 53% (16 of 30) after LVAD were sensitized as measured by SAB (P=0.024). At HTX, 47% (14 of 30) of patients remained sensitized. A positive virtual crossmatch was observed in 28% (4 of 14) of the sensitized patients at HTX. There was no difference between the sensitized and nonsensitized groups (P>0.4 for all) in usage of blood products (6411 vs. 6339 units) and time to HTX (28,663 vs. 25,748 days), and 1 year after HTX, there were no differences in rejection (total rejection score 0.30 vs. 0.37) and survival (93% vs. 88%). Allosensitization after LVAD is common despite cytotoxic PRA being negative. One year after HTX, this sensitization does not translate into increased acute cellular or antibody-mediated rejection or reduced survival.

Costs of Transplantation across a Positive Crossmatch

Purpose There is growing acceptance of the strategy of transplantation across a positive virtual crossmatch (XM) for highly allosensitized pediatric heart transplant (Tx) candidates. While early post-Tx survival is similar to patients (pts) transplanted across a negative XM, the costs of this practice are unknown. We sought to compare hospital charges on the basis of a positive virtual/actual XM. Methods and Materials We reviewed clinical and hospital charge data for all heart Tx recipients at our center from 5/07 to 6/12 (n=60). Charges for all procedures, testing, laboratory studies, therapies, and medications for the Tx admission (day of Tx to hospital discharge) and in the first-year post-Tx (day of Tx to 365d post-Tx in pts with ≥1 yr follow-up) were analyzed. Pts were categorized as virtual/actual XM positive (XM+) if there was pre-Tx donor-specific antibody ≥1000 MFI and/or a positive retrospective XM. Results Median age was 6.2 yrs (15d–20.5 yrs). Pts were 63% male, 32% non-white, and 27% XM+. Diagnosis was cardiomyopathy in 56%, congenital heart disease (CHD) 36%, and re-Tx in 8%. XM+ pts less commonly awaited Tx as inpatients (69 vs. 91%; p=0.048) and more commonly had CHD (81 vs. 32%; p=0.001). Use of plasmapharesis was more common in XM+ pts (50 vs. 5%; p Conclusions Despite the greater use of plasmapharesis, Tx across a positive XM was not associated with longer lengths of stay or higher Tx admission charges. The association with higher first-year charges may reflect increased rejection events, testing/bloodwork, or medication requirements and deserves further study.

Outcomes with High Pre-Transplant PRA in Pediatric Heart Transplantation at a Single Center

Purpose Acceptance and management of pediatric heart transplant (Tx) candidates with high pre-Tx PRA remains center dependent, largely due to lack of medium and longterm followup on outcomes. Some centers proceed only with a negative crossmatch, essentially guaranteeing no suitable donor in the worst cases. Other centers proceed across a positive crossmatch, feeling mortality is more likely waiting for a suitable donor, but with uncertain outcomes. We review outcomes after pediatric heart Tx with positive pre-Tx PRA (>10%) at a center that has not used prospective crossmatch, and more recently has chosen to transplant across a positive virtual crossmatch for those with markedly elevated PRA. Methods and Materials This is a retrospective chart review of all children undergoing heart transplantation at our center. Results We identified 19 recipients with pre-Tx PRA >10%: 11 class I, 2 class II, and 6 both class I and II. PRA class I mean was 43% (range 0-100) and class II mean was 27% (0-100). 11 of 18 tested had a positive retrospective crossmatch; 1 had no crossmatch. Only the 2 most recent recipients had specific intra-operative therapy to reduce antibody. Anti-thymocyte antibody induction is used. Post-Tx medications are often modified for better B cell coverage in this group. There were 5 deaths: rejection 2.3 y; rejection 3.2 y; PTLD 3.4 y; infection 7.0 y; and suicide 10.4 y. Both rejection deaths were linked to noncompliance. None of the high PRA recipients had documented coronary vasculopathy. Actuarial survival was 100% at 1 y; 79% at 5 y; 69% at 10y. 7 of 19 had rejection: 3 cellular, 2 AMR and 2 diagnosed clinically without biopsy. Time to first rejection was 1 y in 3. 3 recipients had >1 rejection episode. Positive crossmatch did not predict those likely to have rejection or death from rejection. Conclusions Reasonable outcomes in pediatric heart Tx recipients with high PRA, with or without a positive crossmatch, support offering Tx across a positive virtual crossmatch for those unlikely to otherwise undergo Tx.

Influence of donor specific HLA antibodies detected by Luminex in kidney graft survival: A multivariate analysis

Some studies have demonstrated the clinical relevance of a positive virtual crossmatch in graft survival; nevertheless, other donor and recipient variables influence the outcome of the transplant. The aim of this study was to investigate the relevance of a positive virtual crossmatch in the graft survival performing a multivariate analysis including other pretransplant variables. A total of 879 deceased kidney transplantations were included. Univariate and multivariate analyses were performed using Cox regression model. After performing the multivariate analysis, a positive virtual crossmatch against class I (adjusted HR 6.613; 95% CI 3.222-13.573), class II (adjusted HR 2.419; 95% CI 1.170-5.002) and class I+II (adjusted HR 5.717; 95% CI 1.925-16.975) detected by single antigen Luminex was the variable conferring the greatest relative risk of graft loss. A positive virtual crossmatch predicts a worse kidney graft survival even after correction by other variables and therefore, transplantation of patients with positive virtual crossmatches should be avoided.

45-P

Aim A negative flow crossmatch (FXM) represents the gold standard for successful kidney transplantation, since this highly sensitive test detects low levels of donor specific antibody (DSA). Positive correlation between FXM and luminex antibody assays is vital for survival of kidney transplant recipients and their grafts. We identified patients eliminated from the UNOS kidney waiting list due to a positive virtual crossmatch, (i.e. DSA levels EXPECTED to yield a positive FXM), yet who actually had a negative FXM result. This enabled our laboratory to develop an improved algorithm which more accurately selects patients’ unacceptable antigens (UA) in UNET. Methods FXMs were performed using sera from 87 potential kidney recipients against 51 different pronase treated (15 min/37 °C) donor cells. This sera was also analyzed for HLA class I and II DSA using luminex single antigen assay (One Lambda). We focused on patient sera containing only one DSA to donor cells tested in the FXM. A correlation between results of the luminex assays (MFI = median fluorescence intensity) and FXM was then determined. Results The average luminex MFI levels for sera exhibiting positive virtual crossmatches and resulting in positive actual FXM was 4,638 ± 379 for class I (n = 89) and 6,684 ± 776 for class II (n = 44) DSA. However, in sera containing DSA expected to produce positive virtual crossmatches but which resulted in negative FXM, the average MFI level was 2,609 ± 142 (n = 60) and 4,824 ± 656 (n = 41) for class I (p Conclusions To reduce false positive predictive results (i.e. virtual crossmatches), luminex MFI cutoffs to select patients’ UA in UNET were increased based on results of actual FXMs. Continual monitoring of correlation between FXM and luminex antibody testing should be performed.

Clinical relevance of HLA donor-specific antibodies detected by single antigen assay in kidney transplantation

Clinical relevance of donor-specific antibodies (DSAs) detected by a single antigen Luminex virtual crossmatch in pre-transplant serum samples from patients with a negative cytotoxicity-dependent complement crossmatch is controversial. The aim of this study was to analyse the influence of a pre-transplant positive virtual crossmatch in the outcome of kidney transplantation. A total of 892 patients who received a graft from deceased donors after a negative cytotoxicity crossmatch were included. Presence of anti-human leucocyte antigen (HLA) antibodies was investigated using a Luminex screening assay and anti-HLA specificities were assigned performing a Luminex single antigen assay. Graft survival was significantly worse among patients with anti-HLA DSA compared to both patients with anti-HLA with no DSA (P = 0.001) and patients without HLA antibodies (P < 0.001) using a log-rank test. No graft survival differences between anti-HLA with no DSA and no HLA antibodies patient groups were observed (P = 0.595). Influence of both anti-Class I and anti-Class II DSA was detected (P < 0.0001 in both cases). When the fluorescence values were stratified in four groups, no significant differences in graft survival were observed among the groups with fluorescence levels >1500 (global P > 0.05). The presence of preformed HLA DSA in transplanted patients with a negative cytotoxicity crossmatch is associated with a lower allograft survival. The detection of anti-HLA with no DSA has no influence in the graft outcome. Finally, there were no demonstrable effects of mean fluorescence intensity (MFI) values >1500 on graft survival.

Positive virtual crossmatch with negative flow crossmatch results in two cases

Pre-transplant (Tx) presence of HLA antibodies (HLA-Ab) especially donor specific antibodies (DSA) has been correlated with post-Tx rejection. While crossmatch (XM) is the specific method to identify DSA, logistical reasons prevent performing a prospective XM in all transplants. In such cases DSA as identified by solid-phase assay (SPA) are being used to perform a virtual crossmatch (VXM). We present two cases, a heart-lung transplant and a kidney transplant, for which testing detected a presumptive DSA with discordant results: a negative flow cytometric crossmatch (FXM) and a positive VXM using SPA. The subsequent investigation determined the antibody, in both cases, was presumably directed against an epitope of a HLA-B*44 antigen found on the single antigen beads (SAB) used in the SPA but not against the native form on the donor lymphocytes used in the FXM. Manufacturing of SAB beads results in denaturation of epitopes, majority of which are removed from the final product, but residual amount is present on the final product. Denaturation of majority of antigen epitopes on single antigen beads did not remove the activity of the recipient's antibodies but it did diminish the activity of positive control serum. This indicates denaturation of some of the HLA-B*44 antigen during manufacturing of the SAB may have lead to the reactivity. Antibody mediated rejection does not appear to be associated with the titer of this antibody to denatured antigen in the first case and so clinical relevance of such antibodies is unclear. Subsequently a second case of discordant FXM and VXM was identified in a potential kidney transplant patient who went on to an uneventful transplant. In this case, lymphocytes from the donor were positively shown to express HLA-B*44:02 using known anti- HLA-B*44:02 control serum. Platelets identified as HLA-B*44:02 could adsorb the anti-HLA-B*44:02 from the control serum activity but not from that of the recipient's anti- HLA-B 44 antibody adding evidence that this antibody should best be classified as a false positive finding. The presence of such an antibody if misidentified may result in unnecessary therapy being instituted or the inappropriate denial of an organ for transplantation.

4-OR: Positive Virtual Crossmatch (VXM), Negative Flow Crossmatch (FXM): A Case of Antibody (Abs) to Denatured Antigen (dAg)

4-OR: Positive Virtual Crossmatch (VXM), Negative Flow Crossmatch (FXM): A Case of Antibody (Abs) to Denatured Antigen (dAg)

Utility of the virtual crossmatch in solid organ transplantation

Solid-phase assays covered with single HLA molecules - such as single-antigen flow-beads (SAFBs) - allow determining the presence of donor-specific HLA antibodies (HLA-DSAs) 'virtually' by comparison of the HLA-antibody specificities of the recipient with the HLA typing of the donor. In this review, prospects and current limitation of the virtual crossmatch are discussed. Several prospective and retrospective studies indicate that a negative virtual crossmatch is associated with a very low risk of early rejection and good long-term allograft survival. By contrast, a positive virtual crossmatch is associated with a significant risk for early rejection and decreased allograft survival. However, these studies revealed that not all HLA-DSAs detected by SAFB have a detrimental clinical impact. The virtual crossmatch has emerged as a very useful tool for pretransplant risk assessment and organ allocation. Further advances of the virtual crossmatch approach will require improvements on the technical part of SAFB analysis and a better understanding and definition of pathogenic factors of HLA-DSA. Together with an extended HLA typing of the donor, this scenario will provide us the full benefits of applications based on virtual crossmatching.

13: Does the Presence of Recipient Antibodies to HLA Antigens or Positive Pre-Transplant Virtual Crossmatch Predict Rejection after Cardiac Transplant?

13: Does the Presence of Recipient Antibodies to HLA Antigens or Positive Pre-Transplant Virtual Crossmatch Predict Rejection after Cardiac Transplant?