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P139 False homozygosity observed for one sample’s HLA genotyping on two independent next generation sequencing platforms
Abstracts / Human Immunology 77 (2016) 40–156
P139
FALSE HOMOZYGOSITY OBSERVED FOR ONE SAMPLE’S HLA GENOTYPING ON TWO INDEPENDENT NEXT GENERATION SEQUENCING PLATFORMS Zahra Kashi, Meagan Barner, Jenefer Dekoning, Gabriel Caceres, RaeAnna Neville, Russ Martin. Kashi Clinical Laboratories, Inc., HLA, Portland, OR, United States. Aim: Next generation sequencing (NGS) was performed on a patient sample to genotype the HLA-DRB1 locus (exons 2 and 3) using both the Ion Torrent PGM and Illumina MiSeq platforms. The sample typed as HLA-DRB1⁄07:01:01 homozygous on both platforms using either the Twin or TypeSteam software analysis packages. Methods: This sample was initially typed by Sequence Specific Oligonucleotide (SSO) hybridization then sequenced using group specific Protrans primers. The SSO and sequencing each typed the sample as 07:01/09:01. Secor reagents were then used to perform Sanger sequencing-based typing (SBT) of exon 2 only. Results: Weak signals were obtained on the SBT forward strand, but the HLA type corroborated the SSO result of 07:01/09:01. Analysis of other samples with 07:01/09:01 allelic combination were found to have genotyped correctly on both NGS platforms. The Staden trev utility program was used to extract Sanger traces from Holotype NGS data. If there were a sufficient number of DRB1⁄09 reads, the Staden trev utility program would have allowed for their detection. Unfortunately, the extracted Sanger traces did not definitively show the presence of DRB1⁄09. The forward Sanger trace showed evidence of DRB1⁄09 in the first 60 bases where there are six ambiguous positions between DRB1⁄07:01 and 09:01. All six positions demonstrated ambiguities supporting the presence of two alleles. However, starting from position 70, signals were weaker and in the eight remaining positions with expected ambiguities, no conclusive signs of DRB1⁄09 were found. Increasing the signal in the utility program allowed for detection of weak ambiguous peaks at all eight of these potentially ambiguous positions. The reverse trace showed stronger support for the presence of DRB1⁄09:01, with the ambiguities post position 70 all in evidence. Discussion: We plan to further sequence the whole DRB1 gene region. This may help illuminate the source of the observed allelic drop out and to rule out mutations that may have impacted the primer binding region for primers designed by two separate manufacturers.
P140
SUCCESSFUL KIDNEY TRANSPLANTATION OF A HIGHLY SENSITIZED PATIENT (HSP) WITH ALLELE-SPECIFIC ANTIBODY (ASA) AGAINST DR52 AFTER SEVERAL REJECTED NATIONAL OFFERS: A CASE REPORT Abubaker M. Sidahmed, Noureddine Berka. University of Calgary, Department of Pathology and Laboratory Medicine and Calgary Laboratory Service, Calgary, AB, Canada. Introduction: The existence of pre-formed donor Specific antibodies (DSA) is considered a contraindication for transplantation. HSPs with cPRA P95% account for about 20% of the waitlist but receive 61% of accessible donor organs. National HSP programs improve the chances of finding donors for these difficult to-match patients but the majority of HSP declined offers are due to ASA not detected by virtual XM. Methods: We present a 54 year old, Caucasian, male, and ABO group A, with ESRD secondary to diabetes mellitus. The patient had a deceased donor (DD) kidney in 1993 that was lost due to chronic rejection and he resumed dialysis in 2004. In October 2006, the patient presented with de-novo class I and class II HLA antibodies. By 2014 the patient’s cPRA was 98% and was listed on the national transplant registry for HSP. Within a period of a year, the patient received 3 HSP offers but were all rejected by the transplant team due to the existence of a strong ASA to HLA DR52. On Oct21 2015, the patient received a virtually negative XM HSP donor offer that was followed with a final negative T cell and weak positive B cells flow XM. The patient was successfully transplanted and in the early outcome he achieved a base line serum creatinine of 126 on day 19 post-surgery and was discharged on Nov9, 2015. On high resolution typing, this patient carries a DRB302:02 allele and has high titre HLA antibodies against DRB301:01 and DRB303:01. We hypothesized that a donor carrying HLA-DRB301:01 will be incompatible to this patient due to the presence of AS DSA. To test this hypothesis, we performed a surrogate XM with a donor that has similar typing to patient except for DRB301:01. This resulted in a T cell negative and B cell positive flow XM. On the other hand, when we used a surrogate donor with DRB302:02 allele, the T and B cell XMs were negative. Discussion: HSP programs have positively impacted the transplantation of the HSP. Since 2004, this patient has had positive B cell flow XMs against more than 30 DDs. Kidney allocation programs as in HSP should include high resolution typing of donors’ HLA and the complete antibody panel screening for the transplant candidate to allow for a safe allocation. HSP programs should flag ASAs to avoid unnecessary offers to patients who are HS and show HLA self reactivity. More data is needed to understand the impact of ASAs on HSP.
139
P139
FALSE HOMOZYGOSITY OBSERVED FOR ONE SAMPLE’S HLA GENOTYPING ON TWO INDEPENDENT NEXT GENERATION SEQUENCING PLATFORMS Zahra Kashi, Meagan Barner, Jenefer Dekoning, Gabriel Caceres, RaeAnna Neville, Russ Martin. Kashi Clinical Laboratories, Inc., HLA, Portland, OR, United States. Aim: Next generation sequencing (NGS) was performed on a patient sample to genotype the HLA-DRB1 locus (exons 2 and 3) using both the Ion Torrent PGM and Illumina MiSeq platforms. The sample typed as HLA-DRB1⁄07:01:01 homozygous on both platforms using either the Twin or TypeSteam software analysis packages. Methods: This sample was initially typed by Sequence Specific Oligonucleotide (SSO) hybridization then sequenced using group specific Protrans primers. The SSO and sequencing each typed the sample as 07:01/09:01. Secor reagents were then used to perform Sanger sequencing-based typing (SBT) of exon 2 only. Results: Weak signals were obtained on the SBT forward strand, but the HLA type corroborated the SSO result of 07:01/09:01. Analysis of other samples with 07:01/09:01 allelic combination were found to have genotyped correctly on both NGS platforms. The Staden trev utility program was used to extract Sanger traces from Holotype NGS data. If there were a sufficient number of DRB1⁄09 reads, the Staden trev utility program would have allowed for their detection. Unfortunately, the extracted Sanger traces did not definitively show the presence of DRB1⁄09. The forward Sanger trace showed evidence of DRB1⁄09 in the first 60 bases where there are six ambiguous positions between DRB1⁄07:01 and 09:01. All six positions demonstrated ambiguities supporting the presence of two alleles. However, starting from position 70, signals were weaker and in the eight remaining positions with expected ambiguities, no conclusive signs of DRB1⁄09 were found. Increasing the signal in the utility program allowed for detection of weak ambiguous peaks at all eight of these potentially ambiguous positions. The reverse trace showed stronger support for the presence of DRB1⁄09:01, with the ambiguities post position 70 all in evidence. Discussion: We plan to further sequence the whole DRB1 gene region. This may help illuminate the source of the observed allelic drop out and to rule out mutations that may have impacted the primer binding region for primers designed by two separate manufacturers.
P140
SUCCESSFUL KIDNEY TRANSPLANTATION OF A HIGHLY SENSITIZED PATIENT (HSP) WITH ALLELE-SPECIFIC ANTIBODY (ASA) AGAINST DR52 AFTER SEVERAL REJECTED NATIONAL OFFERS: A CASE REPORT Abubaker M. Sidahmed, Noureddine Berka. University of Calgary, Department of Pathology and Laboratory Medicine and Calgary Laboratory Service, Calgary, AB, Canada. Introduction: The existence of pre-formed donor Specific antibodies (DSA) is considered a contraindication for transplantation. HSPs with cPRA P95% account for about 20% of the waitlist but receive 61% of accessible donor organs. National HSP programs improve the chances of finding donors for these difficult to-match patients but the majority of HSP declined offers are due to ASA not detected by virtual XM. Methods: We present a 54 year old, Caucasian, male, and ABO group A, with ESRD secondary to diabetes mellitus. The patient had a deceased donor (DD) kidney in 1993 that was lost due to chronic rejection and he resumed dialysis in 2004. In October 2006, the patient presented with de-novo class I and class II HLA antibodies. By 2014 the patient’s cPRA was 98% and was listed on the national transplant registry for HSP. Within a period of a year, the patient received 3 HSP offers but were all rejected by the transplant team due to the existence of a strong ASA to HLA DR52. On Oct21 2015, the patient received a virtually negative XM HSP donor offer that was followed with a final negative T cell and weak positive B cells flow XM. The patient was successfully transplanted and in the early outcome he achieved a base line serum creatinine of 126 on day 19 post-surgery and was discharged on Nov9, 2015. On high resolution typing, this patient carries a DRB302:02 allele and has high titre HLA antibodies against DRB301:01 and DRB303:01. We hypothesized that a donor carrying HLA-DRB301:01 will be incompatible to this patient due to the presence of AS DSA. To test this hypothesis, we performed a surrogate XM with a donor that has similar typing to patient except for DRB301:01. This resulted in a T cell negative and B cell positive flow XM. On the other hand, when we used a surrogate donor with DRB302:02 allele, the T and B cell XMs were negative. Discussion: HSP programs have positively impacted the transplantation of the HSP. Since 2004, this patient has had positive B cell flow XMs against more than 30 DDs. Kidney allocation programs as in HSP should include high resolution typing of donors’ HLA and the complete antibody panel screening for the transplant candidate to allow for a safe allocation. HSP programs should flag ASAs to avoid unnecessary offers to patients who are HS and show HLA self reactivity. More data is needed to understand the impact of ASAs on HSP.
139