Publications (6)3.37 Total impact
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ABSTRACT: Aim NGS is well-suited for HLA typing as it can deliver highly accurate and unambiguous results. A simplified protocol has been developed for use with the Illumina MiSeq and has been subjected to an inter-Methods: Six laboratories, each with varying levels of NGS experience, participated in this double-blinded study. The same 16 samples were typed at HLA-A, B, C, DRB1, and DQB1 by each lab. The protocol consisted of LR-PCR, library prep, and paired-end 250 bp sequencing. Two indexing strategies were employed: (1) Locus-specific indexing by which each locus was tagged uniquely and (2) sample-specific indexing whereby all 5 loci for a sample were pooled prior to library prep. Sequence analysis was performed with Target HLA (Omixon) and NGSengine (GenDx) to assess complementarity of two analysis algorithms. Results Six sequencing runs yielded an average output of 7.8 Gb per run. The average number of sequence reads per library was 387,813. However, analysis was limited to 40K reads for the locus-indexed libraries and 200K reads for the sample-indexed libraries (amplicon pools) resulting in an average coverage of 1444. A sufficient number of reads for genotype analysis were obtained for 98.4% of libraries. Genotype accuracy is shown in Table 1. Reproducibility between labs was 99.7%. The only cause of discordance between labs was cross-contamination of a single amplicon. Conclusion The protocol developed for the MiSeq is reproducible, highly accurate, and its simplicity makes it amenable to clinical testing. The ability of the software to decipher reads from multiple loci in a single library demonstrates the potential for using an amplicon pooling approach that further simplifies sample prep and reduces expense. As demonstrated in this study the protocol is suitable for moderate test volume. However, since <15% of the total reads from each run were needed for analysis it is realistic that >100 samples can be typed in a single run using this approach. A. Berces:Employee; Company/Organization; Omixon, Inc.T. Hague:Employee; Company/Organization; Omixon, Inc.G. Horvath:Consultant; Company/Organization; Omixon, Inc.R. Kooter:Employee; Company/Organization; GenDx. W. Mulder:Stock Shareholder; Company/Organization; GenDx. M. Penning:Employee; Company/Organization; GenDx.K. Rigo:Employee; Company/Organization; Omixon, Inc.E. Rozemuller:Stock Shareholder; Company/Organization; GenDx.Human Immunology. 01/2014; 75:4.
- Nephrology Dialysis Transplantation 02/2008; 23(1):390-2. · 3.37 Impact Factor
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ABSTRACT: In our case series, AMR occurred in patients who had DSA. Twelve of the 21 patients (57%) who developed de novo antibodies post-transplant had biopsy-proven episodes of either rejection (of any Banff classification) and/or chronic allograft nephropathy. Only one-third of these 12 patients with rejection episodes were classified as AMR. It is unclear when and how often measurements for DSA should be performed after transplantation. When AMR is suspected, the detection of anti-HLA antibodies should be done using very sensitive assays. An increase in PRA and/or the detection of anti-HLA antibodies specific to previous sensitization events may precede an episode of AMR, even in the absence of DSA. Prospective clinical trials are needed to assess the predictive value of the presence of DSA after transplant. It is uncertain which therapeutic response should follow after the detection of these antibody responses in the absence of clinical symptoms. AMR requires intensive therapy, but no standard treatment has been established. Three patients who had AMR at our center were treated with rituximab in addition to various combinations of plasmapheresis, methylprednisolone, OKT3, and intravenous immune globulin. Only one patient responded to this treatment. Well-controlled clinical trials would help to evaluate the efficacy and benefit of B-cell depletion in combination with other immunosuppressive agents.Clinical transplants 02/2006;
Article: 71-PHuman Immunology - HUM IMMUNOL. 01/2006; 67.
- Human Immunology - HUM IMMUNOL. 01/2005; 66(8):106-106.
- Human Immunology - HUM IMMUNOL. 01/2004; 65(9).
Hospital of the University of Pennsylvania
Philadelphia, Pennsylvania, United States
- Division of Renal-Electrolyte and Hypertension
University of Pennsylvania
Philadelphia, PA, United States
- Department of Pathology and Laboratory Medicine