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55-OR: Post-Transplant Monitoring: Discordance Between DSA and C4d
S146
Abstracts
55-OR
POST-TRANSPLANT MONITORING: DISCORDANCE BETWEEN DSA AND C4D. Jodi Smith,2 Karen Nelson,1 Paul Warner,2 Danny Youngs,2 Ruth McDonald.1 1Immunogenetics, Puget Sound Blood Center, Seattle, WA, USA; 2Nephrology, Seattle Children’s, Seattle, WA, USA. Aim: HLA Single antigen bead (SAB) assays have increased the sensitivity and specificity of monitoring transplant recipients for donor-specific antibody (DSA). However, not all DSA correlate with C4d staining in biopsies. We examined that correlation in subjects with DSA specific for class II antigens. Methods: Pediatric kidney recipients, negative for DSA pre-transplant, had sera collected at 3 month intervals post-transplant or at biopsy for cause. Sera were tested using single antigen beads, using a threshold of 2000 MFI. Protocol biopsies were performed at 3-6, 12 and 24 months post-transplant, at the time of development of new anti-HLA antibodies, or when clinically indicated. Biopsies were evaluated using Banff criteria and stained for C4d. Results: Twenty-four of 93 subjects (26%) developed de novo DSA. Univariate analysis showed no difference in those developing DSA and those not for the age at transplant, gender, donor source, immunosuppression, or history of acute cellular rejection. Eighteen subjects had DSA to HLA-DQ alone with a mean MFI of 6100 ⫾ 4000. Biopsies from only one of these 18 were positive for C4d and a histology of antibody mediated rejection. However, for the 17/18 negative for C4d, biopsies indicated chronic allograft injury. Four of these 18 had B-cell flow crossmatches performed: 3 were positive. Six subjects developed DSA to both HLA-DR and HLA-DQ. Four of these 6 subjects had histological evidence of antibody mediated rejection and were C4d positive. Conclusions: DSA restricted to HLA-DQ were not predictive of C4d deposition in the kidney allograft in this cohort; the presence of antibodies to HLA-DR increased the incidence of C4d deposition. This may reflect lower expression of HLA-DQ in stable allografts. However, due to the incidence of chronic allograft injury in these subjects, further study of the correlation between DSA specificity and graft function is warranted.
56-OR
COMPARISON BETWEEN TWO DIFFERENT METHODS FOR THE ANALYSIS OF SINGLE ANTIGEN DATA. Chantale Lacelle,1 Ian-Michael Salvador,1 Daniel Hines,1 Lama Hussein,1 Peter Stastny.1,2 1Pathology, UT Southwestern Medical Center, Dallas, TX, USA; 2Internal Medicine - Transplant Immunology, UT Southwestern Medical Center, Dallas, TX, USA. Aim: There is no standard method to analyze Luminex single antigen (SA) data raising some concern that differences in analysis may affect results. We investigated the differences in HLA antibodies detected using either normalized ratios (NR) or a fixed normalized MFI (NMFI) value of 1000. Methods: Class I and II SA data for two consecutive sera from 30 patients were assessed using an in-house analysis which calculates NR and considers NR ⬎2X as positive and the OneLambda software using a fixed 1000 NMFI cutoff. NR were determined by multiplying the raw MFI by an antigen density correction factor and dividing by the mean ⫹3 SD of NHS panel. NMFI values were raw data – NC, minus the serum negative control (LSNC). Results: Antibody specificities identified by NR and not by NMFI were seen in 21/30 (70%) of the patients for class I and 14/30 (47%) of the patients for class II antigens. We found that 81% (17/21) of the patients for whom NR identified additional class I and 57% (8/14) class II specificities and not detected by NMFI, remained positive in subsequent serum samples; while 52% (11/21) of patients negative by NMFI for class I and 36% (5/14) for class II became positive in the later serum. Crossmatches in 2 patients with DSA identified by NR but not NMFI were confirmed as positive in both cases by flow cytometry. Conclusions: Our study shows that the method used to analyze SA tests can impact the antibody specificities identified. NR identified more positive class I and II specificities and yielded more reproducible results when comparing sequential serum sample than NMFI. We also showed that specificities only identified as positive using NR and not by NMFI can cause a positive flow crossmatch.
Abstracts
55-OR
POST-TRANSPLANT MONITORING: DISCORDANCE BETWEEN DSA AND C4D. Jodi Smith,2 Karen Nelson,1 Paul Warner,2 Danny Youngs,2 Ruth McDonald.1 1Immunogenetics, Puget Sound Blood Center, Seattle, WA, USA; 2Nephrology, Seattle Children’s, Seattle, WA, USA. Aim: HLA Single antigen bead (SAB) assays have increased the sensitivity and specificity of monitoring transplant recipients for donor-specific antibody (DSA). However, not all DSA correlate with C4d staining in biopsies. We examined that correlation in subjects with DSA specific for class II antigens. Methods: Pediatric kidney recipients, negative for DSA pre-transplant, had sera collected at 3 month intervals post-transplant or at biopsy for cause. Sera were tested using single antigen beads, using a threshold of 2000 MFI. Protocol biopsies were performed at 3-6, 12 and 24 months post-transplant, at the time of development of new anti-HLA antibodies, or when clinically indicated. Biopsies were evaluated using Banff criteria and stained for C4d. Results: Twenty-four of 93 subjects (26%) developed de novo DSA. Univariate analysis showed no difference in those developing DSA and those not for the age at transplant, gender, donor source, immunosuppression, or history of acute cellular rejection. Eighteen subjects had DSA to HLA-DQ alone with a mean MFI of 6100 ⫾ 4000. Biopsies from only one of these 18 were positive for C4d and a histology of antibody mediated rejection. However, for the 17/18 negative for C4d, biopsies indicated chronic allograft injury. Four of these 18 had B-cell flow crossmatches performed: 3 were positive. Six subjects developed DSA to both HLA-DR and HLA-DQ. Four of these 6 subjects had histological evidence of antibody mediated rejection and were C4d positive. Conclusions: DSA restricted to HLA-DQ were not predictive of C4d deposition in the kidney allograft in this cohort; the presence of antibodies to HLA-DR increased the incidence of C4d deposition. This may reflect lower expression of HLA-DQ in stable allografts. However, due to the incidence of chronic allograft injury in these subjects, further study of the correlation between DSA specificity and graft function is warranted.
56-OR
COMPARISON BETWEEN TWO DIFFERENT METHODS FOR THE ANALYSIS OF SINGLE ANTIGEN DATA. Chantale Lacelle,1 Ian-Michael Salvador,1 Daniel Hines,1 Lama Hussein,1 Peter Stastny.1,2 1Pathology, UT Southwestern Medical Center, Dallas, TX, USA; 2Internal Medicine - Transplant Immunology, UT Southwestern Medical Center, Dallas, TX, USA. Aim: There is no standard method to analyze Luminex single antigen (SA) data raising some concern that differences in analysis may affect results. We investigated the differences in HLA antibodies detected using either normalized ratios (NR) or a fixed normalized MFI (NMFI) value of 1000. Methods: Class I and II SA data for two consecutive sera from 30 patients were assessed using an in-house analysis which calculates NR and considers NR ⬎2X as positive and the OneLambda software using a fixed 1000 NMFI cutoff. NR were determined by multiplying the raw MFI by an antigen density correction factor and dividing by the mean ⫹3 SD of NHS panel. NMFI values were raw data – NC, minus the serum negative control (LSNC). Results: Antibody specificities identified by NR and not by NMFI were seen in 21/30 (70%) of the patients for class I and 14/30 (47%) of the patients for class II antigens. We found that 81% (17/21) of the patients for whom NR identified additional class I and 57% (8/14) class II specificities and not detected by NMFI, remained positive in subsequent serum samples; while 52% (11/21) of patients negative by NMFI for class I and 36% (5/14) for class II became positive in the later serum. Crossmatches in 2 patients with DSA identified by NR but not NMFI were confirmed as positive in both cases by flow cytometry. Conclusions: Our study shows that the method used to analyze SA tests can impact the antibody specificities identified. NR identified more positive class I and II specificities and yielded more reproducible results when comparing sequential serum sample than NMFI. We also showed that specificities only identified as positive using NR and not by NMFI can cause a positive flow crossmatch.