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P150 A case of a shared epitope
Abstracts / Human Immunology 77 (2016) 40–156
P150
A CASE OF A SHARED EPITOPE Reut Hod Dvorai, Nancy D. Herrera, Ashley M. Ruiz, Anat R. Tambur. Northwestern University, Comprehensive Transplant Center, Chicago, IL, United States. A 69-year-old female Caucasian with chronic obstructive pulmonary disease awaiting lung transplant was assessed for her allosensitization status. FlowPRA assay demonstrated significant class II antibodies (0% Class I, 85% Class II) with a strong positive shift suggesting the presence of strong antibodies. In discordance, the single antigen bead (SAB) assay detected only weak Class II antibodies. Patient serum displayed a pan-DR antibody pattern characterized by top-ranking beads representing DR51- and DR52-associated HLA-antigens (550–2590 MFI). As part of our strategy to rule out inhibition, dilution studies were run resulting in a complete diminution of the antibodies observed and indicating absence of inhibition. One limitation of the SAB assay is the fact that it is a multiplex assay. In the presence of an antibody recognizing a target that is present on multiple beads (shared-epitopes), a bead-dependent ‘‘dilution” effect may be observed. This is an alarming scenario as the real strength of an antibody may be missed and the virtual crossmatch (vXM) may not accurately predict the results in a physical XM. However, this limitation can be circumvented by implementing rigorous analysis of specificity patterns and taking into account results from additional assays. In this particular case we chose to run a surrogate Flow cytometric XM (FCXM) to test whether the DR52-associated antigen stacking was a result of epitope-sharing and indicating that the resulting MFI values were an underrepresentation of much stronger reactivity. Surrogate FCXMs were run using two donors. Surrogate [A] was a DR52 homozygous donor (DR12,52) and surrogate [B] was a DR53 homozygous donor (DR4,7,53). The patient was typed as DR1,7,53. FCXM results for surrogate A showed negative T cell and very strong positive B cell, whereas surrogate B had a negative FCXM. This strong positive B cell result was ‘‘unexpected” based on the MFI values associated with the surrogate DR antigens in the SAB assay. Should a transplant have taken place solely based on vXM, the patient may have experienced an aggressive AMR response. This case highlights a limitation of reporting MFI values without rigorous analysis and potentially verifying SAB results by other methodologies. Additional routes to interrogate potential ‘‘epitope-sharing” will be discussed.
P151
ACUTE ANTIBODY-MEDIATED KIDNEY REJECTION ASSOCIATED WITH ANTI-BW6 ORIGINATED FROM ALLOSENSITIZATION TO HLA-C14 DURING PREGNANCY: A CASE REPORT Stephen P. Persaud a, Thalachallour Mohanakumar b, Rowena Delos Santos c, Chang Liu a. aWashington University School of Medicine, Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, St. Louis, MO, United States; bWashington University School of Medicine, Department of Surgery, Saint Louis, MO, United States; cWashington University School of Medicine, Department of Medicine, Division of Nephrology, Saint Louis, MO, United States. We report a case of a 36 year old Caucasian G4P4 female with chronic kidney disease stage 4 and no prior transfusions who received a living-related kidney transplant from her brother, which was a 0A-1B-1DR mismatch. She was discharged post-operative day 4 with good allograft function and a serum creatinine (SCr) of 0.76 mg/dL. Seven days later she presented with pain at the allograft site and a SCr of 3.6 mg/dL. Allograft biopsy showed evidence of thrombotic microangiopathy, hemorrhagic cortical necrosis, and peritubular capillary positivity for C4d. This strongly suggested an acute antibody mediated rejection (AMR), which was later corroborated by histology of the post-nephrectomy allograft specimen. HLA antibody screen at the time of AMR by single-antigen beads demonstrated a broad pattern of reactivity against all beads expressing Bw6associated antigens and multiple HLA-C antigens. The MFI values of all Bw6-associated beads were above 15000 when tested at 1:25 dilution; MFI of the donor specific antibody against B62 (Bw6) was 19366. Only low-level reactivity against a small number of HLA-B antigens was detected prior to transplantation either with neat serum or at 1:25 dilution. These findings are consistent with an anamnestic alloantibody response against a common epitope present in HLA-B and HLA-C antigens, initially primed by exposure to her husband’s antigens during pregnancy and rechallenged by renal transplantation. HLA typing of the patient’s husband demonstrated no antigen mismatch at the A or B loci and one antigen mismatch at the C locus (patient is C16 and her husband is C14). Notably, C14 and all Bw6-associated antigens share a confirmed epitope, 80N (HLA epitope registry). This case demonstrates an unusual incidence of sensitization to Bw6associated antigens via exposure to a Bw6-crossreactive C antigen. It also highlights the risk of occult alloimmunization that is undetectable in pre-transplant HLA antibody testing but may cause rapidly progressive allograft rejection.
147
P150
A CASE OF A SHARED EPITOPE Reut Hod Dvorai, Nancy D. Herrera, Ashley M. Ruiz, Anat R. Tambur. Northwestern University, Comprehensive Transplant Center, Chicago, IL, United States. A 69-year-old female Caucasian with chronic obstructive pulmonary disease awaiting lung transplant was assessed for her allosensitization status. FlowPRA assay demonstrated significant class II antibodies (0% Class I, 85% Class II) with a strong positive shift suggesting the presence of strong antibodies. In discordance, the single antigen bead (SAB) assay detected only weak Class II antibodies. Patient serum displayed a pan-DR antibody pattern characterized by top-ranking beads representing DR51- and DR52-associated HLA-antigens (550–2590 MFI). As part of our strategy to rule out inhibition, dilution studies were run resulting in a complete diminution of the antibodies observed and indicating absence of inhibition. One limitation of the SAB assay is the fact that it is a multiplex assay. In the presence of an antibody recognizing a target that is present on multiple beads (shared-epitopes), a bead-dependent ‘‘dilution” effect may be observed. This is an alarming scenario as the real strength of an antibody may be missed and the virtual crossmatch (vXM) may not accurately predict the results in a physical XM. However, this limitation can be circumvented by implementing rigorous analysis of specificity patterns and taking into account results from additional assays. In this particular case we chose to run a surrogate Flow cytometric XM (FCXM) to test whether the DR52-associated antigen stacking was a result of epitope-sharing and indicating that the resulting MFI values were an underrepresentation of much stronger reactivity. Surrogate FCXMs were run using two donors. Surrogate [A] was a DR52 homozygous donor (DR12,52) and surrogate [B] was a DR53 homozygous donor (DR4,7,53). The patient was typed as DR1,7,53. FCXM results for surrogate A showed negative T cell and very strong positive B cell, whereas surrogate B had a negative FCXM. This strong positive B cell result was ‘‘unexpected” based on the MFI values associated with the surrogate DR antigens in the SAB assay. Should a transplant have taken place solely based on vXM, the patient may have experienced an aggressive AMR response. This case highlights a limitation of reporting MFI values without rigorous analysis and potentially verifying SAB results by other methodologies. Additional routes to interrogate potential ‘‘epitope-sharing” will be discussed.
P151
ACUTE ANTIBODY-MEDIATED KIDNEY REJECTION ASSOCIATED WITH ANTI-BW6 ORIGINATED FROM ALLOSENSITIZATION TO HLA-C14 DURING PREGNANCY: A CASE REPORT Stephen P. Persaud a, Thalachallour Mohanakumar b, Rowena Delos Santos c, Chang Liu a. aWashington University School of Medicine, Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, St. Louis, MO, United States; bWashington University School of Medicine, Department of Surgery, Saint Louis, MO, United States; cWashington University School of Medicine, Department of Medicine, Division of Nephrology, Saint Louis, MO, United States. We report a case of a 36 year old Caucasian G4P4 female with chronic kidney disease stage 4 and no prior transfusions who received a living-related kidney transplant from her brother, which was a 0A-1B-1DR mismatch. She was discharged post-operative day 4 with good allograft function and a serum creatinine (SCr) of 0.76 mg/dL. Seven days later she presented with pain at the allograft site and a SCr of 3.6 mg/dL. Allograft biopsy showed evidence of thrombotic microangiopathy, hemorrhagic cortical necrosis, and peritubular capillary positivity for C4d. This strongly suggested an acute antibody mediated rejection (AMR), which was later corroborated by histology of the post-nephrectomy allograft specimen. HLA antibody screen at the time of AMR by single-antigen beads demonstrated a broad pattern of reactivity against all beads expressing Bw6associated antigens and multiple HLA-C antigens. The MFI values of all Bw6-associated beads were above 15000 when tested at 1:25 dilution; MFI of the donor specific antibody against B62 (Bw6) was 19366. Only low-level reactivity against a small number of HLA-B antigens was detected prior to transplantation either with neat serum or at 1:25 dilution. These findings are consistent with an anamnestic alloantibody response against a common epitope present in HLA-B and HLA-C antigens, initially primed by exposure to her husband’s antigens during pregnancy and rechallenged by renal transplantation. HLA typing of the patient’s husband demonstrated no antigen mismatch at the A or B loci and one antigen mismatch at the C locus (patient is C16 and her husband is C14). Notably, C14 and all Bw6-associated antigens share a confirmed epitope, 80N (HLA epitope registry). This case demonstrates an unusual incidence of sensitization to Bw6associated antigens via exposure to a Bw6-crossreactive C antigen. It also highlights the risk of occult alloimmunization that is undetectable in pre-transplant HLA antibody testing but may cause rapidly progressive allograft rejection.
147