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P036
Abstracts / Human Immunology 75 (2014) 50–141
P036
73
C1Q SINGLE ANTIGEN BEAD ASSAY ONLY DETECTS HIGH TITER/AVIDITY CLASS-I ANTIHLA ANTIBODIES DETECTED BY SINGLE ANTIGEN BEADS. Manish J. Gandhi, Steven DeGoey, Nicole Henderson, Laurie Voit, Justin Kreuter. Mayo Clinic, Rochester, MN, United States. Detection of donor-specific anti-HLA antibodies (DSA) by solid phase assay (SpA) helps donor selection by predicting the actual crossmatch (xM), a process referred as a virtual crossmatch (VxM). At our center we find good correlation (R2 = 0.85) between flow xM (FxM) and VxM using single antigen bead (SAB) assay to identify DSA. The SAB assay is considered a sensitive SpA; however, a concern is that clinically insignificant anti-HLA antibodies (HLA-Ab) may be detected and preclude the recipient from receiving a transplant. The complement fixing (C1q) SAB assay has been proposed as a way to minimize the detection of clinically insignificant HLAAb. We present a summary of our evaluation of this assay. Materials and Methods: 20 positive sera, previously used for serological typing, were tested with SAB, at 1:8 dilution with SAB and by C1q SAB method as per the manufacturer’s protocol. Serological HLA-Ab specificities were compared with those that were obtained by SAB and C1q SAB. For the SpA, specificities >5000 MFI were considered as positive. Specificities were grouped into two categories: 5000–10,000 MFI and >10,000 MFI as in our center >5000 MFI correlates with a positive FxM and >10,000 MFI correlates with cytotoxicity xM. Results: In total there were 53 HLA-Ab specificities as defined by serology, 328 as defined by SAB, and 123 as defined by C1q. SAB identified all 53 specificities defined by serology; 49 with MFI >10,000. Of the 4 with MFI 10,000 on 1:8 dilution and other 2 were not detected by C1q assay. In addition, C1q assay did not detect 3 specificities identified by serology (all with MFI >10,000 by SAB). C1q assay identified 123/179 (69%) of the specificities with MFI >10,000 and 0/151 specificities with MFI: 5000–10,000 as detected by SAB (table). Conclusions: For Class I HLA-Ab, C1q assay only detects high titer/avidity antibodies as defined by SAB. As DSA with SAB MFI: 5000–10,000 can result in a positive FxM, additional investigation is warranted to determine the clinical significance of these HLA-Ab.
Serology positive
SAB Total positive MFI: >10,000 MFI: 5000–10,000 Total negative C1q SAB Total positive MFI: >10,000 MFI: 5000–10,000 Total negative
N
%
53 49 4 0
100 92 8 0
47 47 0 5
89 89 0 11
M.J. Gandhi: Consultant; Company/Organization; Thermo Fisher-One Lambda.
P036
73
C1Q SINGLE ANTIGEN BEAD ASSAY ONLY DETECTS HIGH TITER/AVIDITY CLASS-I ANTIHLA ANTIBODIES DETECTED BY SINGLE ANTIGEN BEADS. Manish J. Gandhi, Steven DeGoey, Nicole Henderson, Laurie Voit, Justin Kreuter. Mayo Clinic, Rochester, MN, United States. Detection of donor-specific anti-HLA antibodies (DSA) by solid phase assay (SpA) helps donor selection by predicting the actual crossmatch (xM), a process referred as a virtual crossmatch (VxM). At our center we find good correlation (R2 = 0.85) between flow xM (FxM) and VxM using single antigen bead (SAB) assay to identify DSA. The SAB assay is considered a sensitive SpA; however, a concern is that clinically insignificant anti-HLA antibodies (HLA-Ab) may be detected and preclude the recipient from receiving a transplant. The complement fixing (C1q) SAB assay has been proposed as a way to minimize the detection of clinically insignificant HLAAb. We present a summary of our evaluation of this assay. Materials and Methods: 20 positive sera, previously used for serological typing, were tested with SAB, at 1:8 dilution with SAB and by C1q SAB method as per the manufacturer’s protocol. Serological HLA-Ab specificities were compared with those that were obtained by SAB and C1q SAB. For the SpA, specificities >5000 MFI were considered as positive. Specificities were grouped into two categories: 5000–10,000 MFI and >10,000 MFI as in our center >5000 MFI correlates with a positive FxM and >10,000 MFI correlates with cytotoxicity xM. Results: In total there were 53 HLA-Ab specificities as defined by serology, 328 as defined by SAB, and 123 as defined by C1q. SAB identified all 53 specificities defined by serology; 49 with MFI >10,000. Of the 4 with MFI 10,000 on 1:8 dilution and other 2 were not detected by C1q assay. In addition, C1q assay did not detect 3 specificities identified by serology (all with MFI >10,000 by SAB). C1q assay identified 123/179 (69%) of the specificities with MFI >10,000 and 0/151 specificities with MFI: 5000–10,000 as detected by SAB (table). Conclusions: For Class I HLA-Ab, C1q assay only detects high titer/avidity antibodies as defined by SAB. As DSA with SAB MFI: 5000–10,000 can result in a positive FxM, additional investigation is warranted to determine the clinical significance of these HLA-Ab.
Serology positive
SAB Total positive MFI: >10,000 MFI: 5000–10,000 Total negative C1q SAB Total positive MFI: >10,000 MFI: 5000–10,000 Total negative
N
%
53 49 4 0
100 92 8 0
47 47 0 5
89 89 0 11
M.J. Gandhi: Consultant; Company/Organization; Thermo Fisher-One Lambda.