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Molecular HLA testing for routine typing of deceased donors
S90
Abstracts
3 100
MOLECULAR HLA TESTING FOR ROUTINE TYPING OF DECEASED DONORS Howard M. Gebel, Lisa Wilmoth-Hosey, Joan E. Holcomb, Pam Chapman, Robert A. Bray, Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA Serological HLA testing for deceased donor work-ups has long been the standard for many laboratories. While serology provides a rapid and cost-effective method to assign HLA antigens, numerous studies have identified the short-comings of serological testing for both Class I and Class II assignments. Nonetheless, serological testing is still considered acceptable since solid-organ allocation is based on HLA antigen (not allele) assignments. In this study, we compared HLA Class I assignments from 250 deceased donors typed both serologically and molecularly (intermediate resolution SSP). Concordant typings were achieved 89% of the time. The discrepancies (28/250; 11%) could be separated into three categories. The first group (N⫽16; 6%) consisted of typings wherein molecular testing identified different HLA antigens than those assigned by serology. All of these differences occured within “broad” HLA antigen groups (ie; HLA-A10, A19 and B15) (e.g., HLA-A*74 vs HLA-A30). The second group (N⫽9; 4%) consisted of molecular typings that identified “splits” of HLA antigens when serology identified only “broad” antigens (e.g., HLA- B*1401/02 vs B14). The final group (N⫽3; 1%) contained two molecular typings that were ambiguous (e.g, HLA-A*03 with *2402/ *3108) but were resolved by serology and one molecular typing that missed a serologically detected antigen (HLA-A36). In the latter situation, the primers were not designed to amplify the rare (A*3603) allele. Collectively, our data show that HLA typing discrepancies are more common with serology. Given the potential impact on both organ allocation and the interpretation of “matching” and outcome, we believe that molecular typing should be performed for all deceased donors.
3 101
USE OF ASSIGN-SBT SOFTWARE FOR QC AND VALIDATION PURPOSES IN SEQUENCING-BASED TYPING Malcolm D. McGinnis,1 Damian Goodridge,2 Jason Stein,1 Pete Krausa,1 David Sayer2, 1Atria Genetics, Inc., South San Francisco, CA, USA; 2Conexio Genomics, East Fremantle, Western Australia Advances in instrumentation and DNA sequencing chemistry have enabled SBT to become a common method for HLA typing. Until recently, a missing component to enable high throughput SBT has been streamlined software analysis. Two critical functions that must be automated are allele typing and quality analysis. Simplified allele typing that minimizes manual editing depends on having proper quality information. Measurements of quality are also essential for determining variation within and between data sets to identify sources of deviation in data quality; e.g. instrumentation, reagent performance, or operator error. Assign-SBT (Conexio Genomics) enables high throughput typing and QC analysis of sequencing data by measuring Quality Values (QV) for each base position in each sequence and provides the tools to analyze the QV scores with statistical outputs. In this study, we used Assign-SBT to perform high resolution typing of AlleleSEQR HLAA, -B, -C, DRB1, DQB1, and DPB1 SBT reagents. As a validation process following a change from DYEnamic ET Terminators (Amersham) to BigDye Terminators (Applied Biosystems), we typed 100 DNA samples from the UCLA DNA Exchange. The panel represented a range of allele types from multiple ethnic origins. The sequence data were analyzed for correct allele types, allele balance (i.e. drop-outs), and sequence quality. The typings were 100% concordant with the consensus results of the UCLA exchange demonstrating the compatibility of the reagent and software combination. Using the Assign-SBT measures of data quality in a routine typing laboratory can help to increase throughput, improve data management, minimize systematic errors, and streamline overall quality control processes.
Abstracts
3 100
MOLECULAR HLA TESTING FOR ROUTINE TYPING OF DECEASED DONORS Howard M. Gebel, Lisa Wilmoth-Hosey, Joan E. Holcomb, Pam Chapman, Robert A. Bray, Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA Serological HLA testing for deceased donor work-ups has long been the standard for many laboratories. While serology provides a rapid and cost-effective method to assign HLA antigens, numerous studies have identified the short-comings of serological testing for both Class I and Class II assignments. Nonetheless, serological testing is still considered acceptable since solid-organ allocation is based on HLA antigen (not allele) assignments. In this study, we compared HLA Class I assignments from 250 deceased donors typed both serologically and molecularly (intermediate resolution SSP). Concordant typings were achieved 89% of the time. The discrepancies (28/250; 11%) could be separated into three categories. The first group (N⫽16; 6%) consisted of typings wherein molecular testing identified different HLA antigens than those assigned by serology. All of these differences occured within “broad” HLA antigen groups (ie; HLA-A10, A19 and B15) (e.g., HLA-A*74 vs HLA-A30). The second group (N⫽9; 4%) consisted of molecular typings that identified “splits” of HLA antigens when serology identified only “broad” antigens (e.g., HLA- B*1401/02 vs B14). The final group (N⫽3; 1%) contained two molecular typings that were ambiguous (e.g, HLA-A*03 with *2402/ *3108) but were resolved by serology and one molecular typing that missed a serologically detected antigen (HLA-A36). In the latter situation, the primers were not designed to amplify the rare (A*3603) allele. Collectively, our data show that HLA typing discrepancies are more common with serology. Given the potential impact on both organ allocation and the interpretation of “matching” and outcome, we believe that molecular typing should be performed for all deceased donors.
3 101
USE OF ASSIGN-SBT SOFTWARE FOR QC AND VALIDATION PURPOSES IN SEQUENCING-BASED TYPING Malcolm D. McGinnis,1 Damian Goodridge,2 Jason Stein,1 Pete Krausa,1 David Sayer2, 1Atria Genetics, Inc., South San Francisco, CA, USA; 2Conexio Genomics, East Fremantle, Western Australia Advances in instrumentation and DNA sequencing chemistry have enabled SBT to become a common method for HLA typing. Until recently, a missing component to enable high throughput SBT has been streamlined software analysis. Two critical functions that must be automated are allele typing and quality analysis. Simplified allele typing that minimizes manual editing depends on having proper quality information. Measurements of quality are also essential for determining variation within and between data sets to identify sources of deviation in data quality; e.g. instrumentation, reagent performance, or operator error. Assign-SBT (Conexio Genomics) enables high throughput typing and QC analysis of sequencing data by measuring Quality Values (QV) for each base position in each sequence and provides the tools to analyze the QV scores with statistical outputs. In this study, we used Assign-SBT to perform high resolution typing of AlleleSEQR HLAA, -B, -C, DRB1, DQB1, and DPB1 SBT reagents. As a validation process following a change from DYEnamic ET Terminators (Amersham) to BigDye Terminators (Applied Biosystems), we typed 100 DNA samples from the UCLA DNA Exchange. The panel represented a range of allele types from multiple ethnic origins. The sequence data were analyzed for correct allele types, allele balance (i.e. drop-outs), and sequence quality. The typings were 100% concordant with the consensus results of the UCLA exchange demonstrating the compatibility of the reagent and software combination. Using the Assign-SBT measures of data quality in a routine typing laboratory can help to increase throughput, improve data management, minimize systematic errors, and streamline overall quality control processes.