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P044 One year experience with HLA typing by next generation sequencing (NGS), achievements and challenges
Abstracts / Human Immunology 78 (2017) 51–254
P044
ONE YEAR EXPERIENCE WITH HLA TYPING BY NEXT GENERATION SEQUENCING (NGS), ACHIEVEMENTS AND CHALLENGES Ana Hernandez, Jennifer McCue, Emilio Margolles-Clark, Phillip Ruiz. University of Miami, Miami, FL, United States. Aim: After a year of implementing HLA typing by NGS, we present our most relevant achievements and challenges. Methods: Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLADQ, and Exon 2 thru 3’UTR of HLA-DP) amplified with NXTypeTM Class I and Class II primer sets. Automated template preparation and chip loading were performed in the Ion ChefTM system and run on Ion S5TM. Data was analyzed with TypeStreamTM v1.1.0.11 software. Results: Having typed over 1000 samples by NGS, with 50% from bone marrow programs and 50% from solid organ programs, our workflow has changed significantly: Threefold increase in capacity with simultaneous decrease in turn-around-time (TAT, on average 10 days) for high resolution typing. Our current capacity is up to 96 HLA typings in 3–4 days vs. minimum of 16 days for the same volume with SBT by Sanger. Resolution of most ambiguities (98.7%), minimizing the need for reflex testing.Repetitions due to technical failures and ambiguities decreased from 4.3% with SBT-Sanger to 0.3% with NGS. In spite of positive workflow changes, there remain challenges: Automation of library preparation has not been implemented.No merging of library preparation reagents into a single kit.Smaller runs increase the number of reads, leading to artifacts and high background generated by the software, interfering with analysis and limiting flexibility for STAT sample processing.For solid organ typings, generating high resolution results while reporting Serological Equivalents for UNOS, poses logistical difficulties.TypeStreamTM software not interfaced with Fusion, requiring continuous need to run SSOP for DSA analysis.Unresolved ambiguities (1.3%), due to incomplete exon coverage and phase ambiguities. Resolution may be obtained by increasing gene coverage, length of reads and with additional testing methods. Conclusions: We have confirmed that NGS is both a suitable and faster method to obtain high resolution HLA typing. We have seen a significant decrease in TAT along with a significant cost savings due to fewer repetitions, fewer reagents, and most significantly, reduced time from our most valuable asset, our technologists.
P045
COMPLEMENT BINDING TO SINGLE ANTIGEN BEADS IS NOT DIRECTLY RELATED TO ANTIBODY STRENGTH AS DETERMINED IN PAIRWISE COMPARATIVE STUDIES Allen J. Norin, Ballabh Das, David Hochman, Devon John, Nabil Sumrani, Moro O. Salifu. SUNY Downstate Medical Center, Brooklyn, NY, United States. Aim: Optical bead microarrays using Luminex have largely replaced anti-HLA antibody screening tests using cell based complement dependent cytotoxicity assays. One unintended consequence of this technologic development was the loss of the ability to distinguish between antibodies that fix complement from those that do not. This situation may have been addressed by the introduction of several assays that use the bead microarrays in conjunction with a reagent that detects whether components of the complement cascade are bound to the anti-HLA antibody – HLA complex on a reactive bead. One controversy centering on this technology has been the suggestion that the binding of complement in bead assays is dependent to a greater extend on the strength of the anti-HLA antibody rather than its complement binding activity. To address this issue we examined complement binding in single antigen bead (SAB) assays in sera that had multiple epitope specificities of different strengths. Methods: Patient sera and sera from proficiency surveys were used. SAB assays of these sera were compared to C1q/C3d – SAB assays. The bead with the highest net MFI for each identified epitope in the microarray was recorded and compared to the same bead in C1q/C3d – SAB assays. Results: We detected both strong and weak anti-HLA antibodies in SAB assay that were detected in C1q/ C3d – SAB assays. In some sera complement bound to one set of epitope reactive SABs but not to another epitope set of SABs. Importantly, this was not dependent on the strength of the antibody. For example, in one patient’s sera, an epitope defined antigen bead, B⁄ 42:01 (epitope 65GK), had an MFI of 12,989 (normalized score) in the SAB assay but only 99 MFI in the C1q – SAB assay. In the same serum another epitope defined bead (80 N), A⁄ 23:01 with an MFI of 10,445 in the SAB assay exhibited an MFI of 9,162 in the C1q – SAB assay. We also observed the latter finding for HLA class 2 SABs and with the C3d assay. Conclusions: Complement binding to SABs is not directly related to strength of the antibody as determined in pairwise comparative studies using sera that contained antibodies with different epitope specificities. C1q/ C3d - SAB assays are therefore useful in evaluating the complement binding properties of pretransplant and post transplant anti-HLA antibodies.
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P044
ONE YEAR EXPERIENCE WITH HLA TYPING BY NEXT GENERATION SEQUENCING (NGS), ACHIEVEMENTS AND CHALLENGES Ana Hernandez, Jennifer McCue, Emilio Margolles-Clark, Phillip Ruiz. University of Miami, Miami, FL, United States. Aim: After a year of implementing HLA typing by NGS, we present our most relevant achievements and challenges. Methods: Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLADQ, and Exon 2 thru 3’UTR of HLA-DP) amplified with NXTypeTM Class I and Class II primer sets. Automated template preparation and chip loading were performed in the Ion ChefTM system and run on Ion S5TM. Data was analyzed with TypeStreamTM v1.1.0.11 software. Results: Having typed over 1000 samples by NGS, with 50% from bone marrow programs and 50% from solid organ programs, our workflow has changed significantly: Threefold increase in capacity with simultaneous decrease in turn-around-time (TAT, on average 10 days) for high resolution typing. Our current capacity is up to 96 HLA typings in 3–4 days vs. minimum of 16 days for the same volume with SBT by Sanger. Resolution of most ambiguities (98.7%), minimizing the need for reflex testing.Repetitions due to technical failures and ambiguities decreased from 4.3% with SBT-Sanger to 0.3% with NGS. In spite of positive workflow changes, there remain challenges: Automation of library preparation has not been implemented.No merging of library preparation reagents into a single kit.Smaller runs increase the number of reads, leading to artifacts and high background generated by the software, interfering with analysis and limiting flexibility for STAT sample processing.For solid organ typings, generating high resolution results while reporting Serological Equivalents for UNOS, poses logistical difficulties.TypeStreamTM software not interfaced with Fusion, requiring continuous need to run SSOP for DSA analysis.Unresolved ambiguities (1.3%), due to incomplete exon coverage and phase ambiguities. Resolution may be obtained by increasing gene coverage, length of reads and with additional testing methods. Conclusions: We have confirmed that NGS is both a suitable and faster method to obtain high resolution HLA typing. We have seen a significant decrease in TAT along with a significant cost savings due to fewer repetitions, fewer reagents, and most significantly, reduced time from our most valuable asset, our technologists.
P045
COMPLEMENT BINDING TO SINGLE ANTIGEN BEADS IS NOT DIRECTLY RELATED TO ANTIBODY STRENGTH AS DETERMINED IN PAIRWISE COMPARATIVE STUDIES Allen J. Norin, Ballabh Das, David Hochman, Devon John, Nabil Sumrani, Moro O. Salifu. SUNY Downstate Medical Center, Brooklyn, NY, United States. Aim: Optical bead microarrays using Luminex have largely replaced anti-HLA antibody screening tests using cell based complement dependent cytotoxicity assays. One unintended consequence of this technologic development was the loss of the ability to distinguish between antibodies that fix complement from those that do not. This situation may have been addressed by the introduction of several assays that use the bead microarrays in conjunction with a reagent that detects whether components of the complement cascade are bound to the anti-HLA antibody – HLA complex on a reactive bead. One controversy centering on this technology has been the suggestion that the binding of complement in bead assays is dependent to a greater extend on the strength of the anti-HLA antibody rather than its complement binding activity. To address this issue we examined complement binding in single antigen bead (SAB) assays in sera that had multiple epitope specificities of different strengths. Methods: Patient sera and sera from proficiency surveys were used. SAB assays of these sera were compared to C1q/C3d – SAB assays. The bead with the highest net MFI for each identified epitope in the microarray was recorded and compared to the same bead in C1q/C3d – SAB assays. Results: We detected both strong and weak anti-HLA antibodies in SAB assay that were detected in C1q/ C3d – SAB assays. In some sera complement bound to one set of epitope reactive SABs but not to another epitope set of SABs. Importantly, this was not dependent on the strength of the antibody. For example, in one patient’s sera, an epitope defined antigen bead, B⁄ 42:01 (epitope 65GK), had an MFI of 12,989 (normalized score) in the SAB assay but only 99 MFI in the C1q – SAB assay. In the same serum another epitope defined bead (80 N), A⁄ 23:01 with an MFI of 10,445 in the SAB assay exhibited an MFI of 9,162 in the C1q – SAB assay. We also observed the latter finding for HLA class 2 SABs and with the C3d assay. Conclusions: Complement binding to SABs is not directly related to strength of the antibody as determined in pairwise comparative studies using sera that contained antibodies with different epitope specificities. C1q/ C3d - SAB assays are therefore useful in evaluating the complement binding properties of pretransplant and post transplant anti-HLA antibodies.
87