Jar-How Lee

Denatured class I human leukocyte antigen antibodies in sensitized kidney recipients: prevalence, relevance, and impact on organ allocation.

Background Single antigen flow beads assays may overestimate sensitization because of the detection of supposedly irrelevant antibodies recognizing denatured class I human leukocyte antigens (HLAs). Methods Sera of 323 HLA-sensitized kidney transplant candidates positive with a class I HLA single antigen flow beads assay were retested after acid treatment of the beads. Denatured HLA antibodies were identified according to ratio between the measured fluorescence intensity for treated and nontreated beads. T-lymphocyte flow cytometry crossmatches were performed to characterize the ability of these antibodies to recognize HLA on normal cells as a surrogate of their potential clinical relevance. Their impact on organ allocation was evaluated through a calculated panel reactive antibody. The utility of single antigen flow beads largely devoid of denatured HLA (iBeads) was also evaluated. Results Denatured HLA antibodies were detected in 39% of the patients. They provided much less positive flow cytometry crossmatches than anti-native HLA antibodies (16% vs. 83%, P<0.0001). Removing the HLA-A and HLA-B antigens targeted by denatured HLA antibodies from unacceptable antigens lowered the calculated panel reactive antibody for 90 patients, sometimes dramatically. The iBeads assay demonstrated nearly the same ability to predict crossmatch results than the acid treatment assay. Conclusion Denatured class I HLA antibodies are common, but the antigens they target should not be considered as unacceptable in most cases, because they negatively impact access to a transplant while predominantly providing negative sensitive crossmatches. The iBeads assay seems to be a valuable alternative to better define unacceptable antigens.

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation

Anti‐HLA donor‐specific antibodies are deleterious for organ transplant survival. Class I HLA donor‐specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti‐denatured HLA antibodies (anti‐dHLAs). Anti‐dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti‐dHLAs to be discriminated from anti‐nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid‐treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti‐dHLAs. However, some anti‐dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti‐nHLAs conserved significant reactivity toward acid‐treated LSAB. After depleting serum anti‐nHLA reactivity with HLA‐typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid‐treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti‐nHLAs and anti‐dHLAs, or anti‐nHLAs recognizing acid‐resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti‐HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti‐HLA antibodies in human serum.

Clinical impact of preformed donor-specific denatured class I HLA antibodies after kidney transplantation.

Class I single‐antigen flow beads (SAFB) carry native and denatured human leukocyte antigen (HLA) molecules. Using a cohort of 179 class I HLA‐sensitized kidney recipients, we described incidence and clinical relevance of preformed denatured HLA donor‐specific antibodies (DSA) using two different assays: an acid‐treated SAFB assay (anti‐dHLA DSA) and the iBeads assays (SAFB+/iBeads‐ DSA). Eighty‐five class I DSA were found in 67 patients (median mean fluorescence intensity [MFI] of 1729 [range 520–13 882]). Anti‐dHLA and SAFB+/iBeads‐ DSA represented 11% and 18% of class I DSA and were mainly low MFI DSA (500–1000 MFI). Concordance between these two assays was good (90%). None of the patients with only class I anti‐dHLA DSA or only SAFB+/iBeads‐ DSA developed acute clinical antibody‐mediated rejection in the first‐year post‐transplantation, and their five‐yr death‐censored graft survival was similar to that of patients without DSA. Moreover, all these patients displayed a negative current T‐cell flow cytometry cross‐match. Therefore, both anti‐dHLA DSA and SAFB+/iBeads‐ DSA appear irrelevant, which could explain the good outcome observed in some patients with preformed class I DSA.

Evaluation of the iBeads assay as a tool for identifying class I HLA antibodies

In addition to antibodies targeting native class I human leukocyte antigens (HLA), the single antigen flow beads assay (SAFB) detects antibodies recognizing denatured forms (anti-dHLA). Acid treated SAFB and the modified SAFB reagent named iBeads are expected to distinguish anti-native (anti-nHLA) from anti-dHLA. Sera from 280 class I HLA-sensitized SAFB-positive kidney transplant candidates were retested with acid-treated SAFB and iBeads. Concordance between SAFB and iBeads, taking into account acid-treatment results, was described at global and locus levels. T-lymphocyte flow cytometry crossmatches (FCXM) were performed to identify an accurate iBeads MFI threshold allowing predicting FCXM results. Concordance between acid-treatment and iBeads assays was observed for 86.9% of alleles. The iBeads MFI were lower than for classical SAFB, especially for HLA-B and C alleles. Anti-dHLA identified with acid-treated SAFB were more frequently negative with iBeads for HLA-B and -C alleles. An iBeads MFI threshold of 1000 allowed predicting positive FCXM with 95.6% sensitivity, 91.6% negative predictive value and 0.08 negative likelihood ratio. The iBeads assay still has limitations, but might represent an invaluable alternative to SAFB for virtual crossmatch strategies in organ transplant allocation programs.

Inter and intra laboratory concordance of HLA antibody results obtained by single antigen bead based assay.

Single antigen bead based assays (SAB) to identify antibodies to HLA are marketed as a qualitative test, however often used as a quantitative test as the results provide mean fluorescence intensity (MFI) which is found to correlate with the strength/avidity of the antibody. We studied the between and within laboratory variability in performing the SAB from one manufacturer. Ten samples were tested at four laboratories according to the manufacturers suggested protocol. Additionally same samples were tested on four consecutive days in one laboratory. All tests were performed using the same lot of beads and secondary antibody. Results were classified as positive at four different MFI cutoffs: 1000, 5000, 8000 and 10,000. MFI values across and within the laboratory were compared in a pair-wise fashion with Pearsons correlations. Overall concordance for Class-I was 97% between laboratories and 98% within laboratory at all cutoffs. Pair-wise Pearson correlation between laboratories was 0.989-0.99, while within laboratory it was 0.998-0.999. For Class-II, overall concordance between and within laboratory was 98%. Pair-wise Pearson correlation between laboratories was 0.991-0.997, while within laboratory it was 0.997-0.999. There is good correlation between laboratories and within laboratory using the same manufacturer, same lot and same protocol while performing SAB.

Reassessment of T Lymphocytes Crossmatches Results Prediction With Luminex Class I Single Antigen Flow Beads Assay.

Background In virtual crossmatch (XM) strategies, a correct anticipation of XM results is required for appropriately allocating organs. We reassessed the ability to predict T lymphocyte flow cytometry and complement dependent cytotoxicity XM results with the mean fluorescence intensity (MFI) in Luminex class I single antigen flow beads (SAFB) assay, after correction of complement interference and exclusion of antidenatured HLA antibodies. Methods Among 432 XM with T lymphocytes (T-XM), 407 were analyzed after exclusion of antidenatured HLA antibodies. Only ethylenediaminetetraacetic acid-treated serum MFI was considered. Only 1 cellular target HLA antigen for the serum was expressed in 238 cases, 209 and 29 being heterozygous and homozygous for this antigen, respectively. For the remaining 169 cases, at least 2 antigens were recognized. Single antigen flow bead MFI thresholds allowing XM positivity to be predicted were calculated with receiver operating characteristic curves. Results T-XM results were tightly associated with SAFB MFI values. Anti-HLA-A and anti-HLA-B antibodies behaved similarly. Prediction and sensitivity SAFB MFI thresholds were determined, respectively, assessing the highest sensitivity/specificity ratio and at least 95% sensitivity for predicting T-XM positivity. Both were slightly lower for HLA-B than for HLA-A, whereas anti-HLA-Cw antibodies induced random XM results. Both thresholds were only slightly diminished for homozygous and for multiple HLA targets, considering the immunodominant, but not the sum, of antibodies MFI. Conclusions Antibodies against HLA-A, -B, or -Cw behave differently. A homozygous HLA target does not trigger a twice higher XM positivity. Multiple antibodies are better evaluated through the immunodominant DSA MFI than through the sum of DSA MFI.

Mapping molecular HLA typing data to UNOS antigen equivalents for improved virtual crossmatch

Background Virtual crossmatch utilizes HLA typing and antibody screen assay data as a part of organ offers in deceased donor allocation systems. Histocompatibility labs must convert molecular HLA typings to antigen equivalencies for entry into the United Network for Organ Sharing (UNOS) UNet system. While an Organ Procurement and Transplantation Network (OPTN) policy document provides general guidelines for conversion, the process is complex because no antigen mapping table is available. We present a UNOS antigen equivalency table for all IMGT/HLA alleles at the A, B, C, DRB1, DRB3/4/5, DQA1, and DQB1 loci. Methods An automated script was developed to generate a UNOS antigen equivalency table. Data sources used in the conversion algorithm included the World Marrow Donor Association(WMDA) antigen table, the HLA Dictionary, and UNOS-provided tables. To validate antigen mappings, we converted National Marrow Donor Program (NMDP) high resolution allele frequencies to antigen equivalents and compared with the UNOS Calculated Panel Reactive Antibodies (CPRA) reference panel. Results Normalized frequency similarity scores between independent NMDP and UNOS panels for 4 US population categories (Caucasian, Hispanic, African American and Asian/Pacific Islander) ranged from 0.85 to 0.97, indicating correct antigen mapping. An open source web application (ALLele to ANtigen (“ALLAN”)) and web services were also developed to map unambiguous and ambiguous HLA typing data to UNOS antigen equivalents based on NMDP population-specific allele frequencies (http://www.transplanttoolbox.org). Conclusions This tool sets a foundation for using molecular HLA typing to compute the virtual crossmatch and may aid in reducing typing discrepancies in UNet.