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P114 Validation of high resolution HLA typing using NXTypeTM Ion-Torrent next generation sequencing. The University of Miami experience
Abstracts / Human Immunology 77 (2016) 40–156
P113
PREDICTIVE VALUE OF C1Q DONOR SPECIFIC ANTIBODY IN IDENTIFYING PATHOLOGIC FEATURES OF AMR Elizabeth H. Field a,b, Sarat Kuppachi a, Diana Zepada-Orozco c, Danniele G. Holanda d. aUniversity of Iowa, Internal Medicine, Iowa City, IA, United States; bVeteran Affairs Medical Center, Iowa City, IA, United States; c University of Iowa, Pediatrics, Iowa City, IA, United States; dUniversity of Iowa, Pathology, Iowa City, IA, United States. Aims: Donor-specific antibody (DSA) correlates with antibody-mediated rejection (AMR), transplant glomerulopathy and graft loss. However, the threshold of when to treat DSA remains a clinical challenge, as it is not clear whether earlier intervention in the AMR process will improve the success of persistently reducing DSA and prolonging outcomes. To address the need for enhanced recognition of AMR diagnosis, we evaluated the predictive value of C1q and conventional IgG DSA levels in identifying AMR features on biopsy. Methods: We performed a retrospective quality control study of 39 biopsies from 20 patients with stored serum samples near the time of biopsy. Serum was tested for DSA by C1q using One Lambda SA reagents modified with anti-C1q-biotin plus streptavidin-PE and by conventional IgG. Biopsies were reviewed by a renal pathologist and renal transplant nephrologist who were blinded to immunologic data, and pathologic features of AMR were extracted and classified. Fisher Exact Test was used on categorical data to test significance of correlations between DSA tests and AMR features. Positive and Negative predictive values (PPV, NPV), sensitivity and specificity of IgG DSA and C1q DSA were calculated for different MFI cut-offs. Results: IgG DSA correlated with positive C4d at MFI over 1300 (p < .003) but had poor PPV because of false positive cases of AMR without C4d. C1q DSA correlated with positive C4d on biopsy with cut-off values at or above 150 MFI (p < .023), with cut-off of 700 MFI (p < .001) performing the best (PPV 88.9%, NPV 93.1%, sensitivity 80%, specificity 96.4%). In contrast, IgG DSA correlated with one or more features of AMR at MFI over 1300 (p < .001, PPV 83.3%, NPV 75%, sensitivity 75%, specificity 83.3%), but C1q DSA did not. Using combined MFI values of the four highest DSAs and MFI cutoffs of either IgG >4500 or C1q >600 correlated with and predicted presence of one or more biopsy feature of AMR (p < .001, PPV 90%, NPV 84.2%, sensitivity 85.7%, specificity 88.9%) and identified all cases with C4d staining. Conclusion: Incorporating both IgG and C1q DSA results criteria improved predictability of AMR features on biopsy. These may be useful criteria for establishing earlier pathological or clinical diagnosis of AMR and for developing treatment decision algorithms.
P114
VALIDATION OF HIGH RESOLUTION HLA TYPING USING NXTYPETM ION-TORRENT NEXT GENERATION SEQUENCING. THE UNIVERSITY OF MIAMI EXPERIENCE Dania Mateu, Emilio Margolles-Clark, Ana Hernandez, Phillip Ruiz. University of Miami, Surgery. Transplant Laboratory, Miami, FL, United States. Aim: HLA typing using next generation sequencing (NGS) takes the Sanger based sequencing HLA typing to a higher dimension. The technology permits multiplexing of several target regions and various samples in a single run. The advantages of this technology include high throughput, improved DNA sequencing accuracy and faster data analysis. Our aim was validate the NXTypeTM Ion-Torrent NGS HLA typing procedure to implement it in our clinical laboratory. Methods: 214 samples were analyzed with NXTypeTM Ion-Torrent NGS and compared to high resolution typing obtained by Sanger sequencing. HLA Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLA-DQ, and Exon 2 thru 3’UTR of HLA-DP) were amplified with NXTypeTM Class I and Class II primers set, respectively. Amplicons were fragmented with Ion ShearTM Plus Reagents Kit to sizes from 350 to 950bp. Libraries were enriched in OneTouch ES and run in an Ion PGMTM. Data was analyzed with HLATypeStream (v1.0.0.86) software. Results: We obtained good coverage of the target regions when multiplexing 24 samples per run using a 318TM v2 chip. Of the 1070 alleles analyzed (214 samples), 143 were homozygous and 927 heterozygous. The concordance between Sanger sequencing results and NGS was 99.2%, 99.6%, 99.8%, 100% and 99.8% for loci A, B, C, DRB1 and DQB1, respectively. The number of different alleles identified for loci A, B, C, DRB1 and DQB1 were 40, 69, 36, 42, and 18, respectively. Our results revealed that 269 NMDP codes reported by Sanger sequencing were resolved by NGS, except 3 codes in DRB1 because of mismatches present in exons 1 or 4, which are not targeted by NXTypeTM Class II primers. We found two new alleles in exons (A⁄24, exon 1 and C⁄01, exon 2) and variations in intronic regions of 46 alleles. All null alleles A⁄68:11N (10), B⁄15:01N (11) and C⁄04:09N/B⁄44:03 (3) seen by Sanger were resolved by NGS. Conclusions: The NGS is a reliable and accurate method to determine HLA typing of a considerable number of samples relatively fast, solving most allele ambiguities (98.7%) without the need of alternative tests. We confirmed that NXTypeTM Ion-Torrent NGS is a suitable and quick method for routine use in clinical laboratories to obtain complete ultra-high-resolution HLA typing that ultimately will improve on current HLA matching for transplant.
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P113
PREDICTIVE VALUE OF C1Q DONOR SPECIFIC ANTIBODY IN IDENTIFYING PATHOLOGIC FEATURES OF AMR Elizabeth H. Field a,b, Sarat Kuppachi a, Diana Zepada-Orozco c, Danniele G. Holanda d. aUniversity of Iowa, Internal Medicine, Iowa City, IA, United States; bVeteran Affairs Medical Center, Iowa City, IA, United States; c University of Iowa, Pediatrics, Iowa City, IA, United States; dUniversity of Iowa, Pathology, Iowa City, IA, United States. Aims: Donor-specific antibody (DSA) correlates with antibody-mediated rejection (AMR), transplant glomerulopathy and graft loss. However, the threshold of when to treat DSA remains a clinical challenge, as it is not clear whether earlier intervention in the AMR process will improve the success of persistently reducing DSA and prolonging outcomes. To address the need for enhanced recognition of AMR diagnosis, we evaluated the predictive value of C1q and conventional IgG DSA levels in identifying AMR features on biopsy. Methods: We performed a retrospective quality control study of 39 biopsies from 20 patients with stored serum samples near the time of biopsy. Serum was tested for DSA by C1q using One Lambda SA reagents modified with anti-C1q-biotin plus streptavidin-PE and by conventional IgG. Biopsies were reviewed by a renal pathologist and renal transplant nephrologist who were blinded to immunologic data, and pathologic features of AMR were extracted and classified. Fisher Exact Test was used on categorical data to test significance of correlations between DSA tests and AMR features. Positive and Negative predictive values (PPV, NPV), sensitivity and specificity of IgG DSA and C1q DSA were calculated for different MFI cut-offs. Results: IgG DSA correlated with positive C4d at MFI over 1300 (p < .003) but had poor PPV because of false positive cases of AMR without C4d. C1q DSA correlated with positive C4d on biopsy with cut-off values at or above 150 MFI (p < .023), with cut-off of 700 MFI (p < .001) performing the best (PPV 88.9%, NPV 93.1%, sensitivity 80%, specificity 96.4%). In contrast, IgG DSA correlated with one or more features of AMR at MFI over 1300 (p < .001, PPV 83.3%, NPV 75%, sensitivity 75%, specificity 83.3%), but C1q DSA did not. Using combined MFI values of the four highest DSAs and MFI cutoffs of either IgG >4500 or C1q >600 correlated with and predicted presence of one or more biopsy feature of AMR (p < .001, PPV 90%, NPV 84.2%, sensitivity 85.7%, specificity 88.9%) and identified all cases with C4d staining. Conclusion: Incorporating both IgG and C1q DSA results criteria improved predictability of AMR features on biopsy. These may be useful criteria for establishing earlier pathological or clinical diagnosis of AMR and for developing treatment decision algorithms.
P114
VALIDATION OF HIGH RESOLUTION HLA TYPING USING NXTYPETM ION-TORRENT NEXT GENERATION SEQUENCING. THE UNIVERSITY OF MIAMI EXPERIENCE Dania Mateu, Emilio Margolles-Clark, Ana Hernandez, Phillip Ruiz. University of Miami, Surgery. Transplant Laboratory, Miami, FL, United States. Aim: HLA typing using next generation sequencing (NGS) takes the Sanger based sequencing HLA typing to a higher dimension. The technology permits multiplexing of several target regions and various samples in a single run. The advantages of this technology include high throughput, improved DNA sequencing accuracy and faster data analysis. Our aim was validate the NXTypeTM Ion-Torrent NGS HLA typing procedure to implement it in our clinical laboratory. Methods: 214 samples were analyzed with NXTypeTM Ion-Torrent NGS and compared to high resolution typing obtained by Sanger sequencing. HLA Class I (full HLA-A, -B, and -C genes) and HLA Class II (Exon 2 thru intron 3 of HLA-DR and HLA-DQ, and Exon 2 thru 3’UTR of HLA-DP) were amplified with NXTypeTM Class I and Class II primers set, respectively. Amplicons were fragmented with Ion ShearTM Plus Reagents Kit to sizes from 350 to 950bp. Libraries were enriched in OneTouch ES and run in an Ion PGMTM. Data was analyzed with HLATypeStream (v1.0.0.86) software. Results: We obtained good coverage of the target regions when multiplexing 24 samples per run using a 318TM v2 chip. Of the 1070 alleles analyzed (214 samples), 143 were homozygous and 927 heterozygous. The concordance between Sanger sequencing results and NGS was 99.2%, 99.6%, 99.8%, 100% and 99.8% for loci A, B, C, DRB1 and DQB1, respectively. The number of different alleles identified for loci A, B, C, DRB1 and DQB1 were 40, 69, 36, 42, and 18, respectively. Our results revealed that 269 NMDP codes reported by Sanger sequencing were resolved by NGS, except 3 codes in DRB1 because of mismatches present in exons 1 or 4, which are not targeted by NXTypeTM Class II primers. We found two new alleles in exons (A⁄24, exon 1 and C⁄01, exon 2) and variations in intronic regions of 46 alleles. All null alleles A⁄68:11N (10), B⁄15:01N (11) and C⁄04:09N/B⁄44:03 (3) seen by Sanger were resolved by NGS. Conclusions: The NGS is a reliable and accurate method to determine HLA typing of a considerable number of samples relatively fast, solving most allele ambiguities (98.7%) without the need of alternative tests. We confirmed that NXTypeTM Ion-Torrent NGS is a suitable and quick method for routine use in clinical laboratories to obtain complete ultra-high-resolution HLA typing that ultimately will improve on current HLA matching for transplant.
121