- Email: [email protected]
Evalution of electronic dp epitype profiling to determine dp donor-specific antibodies during virtual crossmatch
176
P152
Abstracts / Human Immunology 79 (2018) 58–187
EVALUTION OF ELECTRONIC DP EPITYPE PROFILING TO DETERMINE DP DONOR-SPECIFIC ANTIBODIES DURING VIRTUAL CROSSMATCH Dong-Feng Chen 1, Wendy E. Hanshew 1, Divyank Saini 2, Barbara Ure 2, Collin Brack 2. 1 Duke University Medical Center, Durham, NC, United States; 2 HLA Data Systems, Houston, TX, United States. Aim: Our aim is to evaluate the utility of incorporating epitype analysis into the computerized virtual crossmatch (vXM) workflow for donors with DP alleles that are not covered by a recipient’s HLA antibody screening and identification assay. Our lab has used vXM to select deceased donors for transplantations for more than ten years. If the donor DP antigens are covered by the assay, the DSA can be determined directly by vXM. If the donor antigen is not included in the assay but is covered by an epitype (EDP) group which includes a DP antigen covered by the assay, the DSA can be determined based on the positivity of the EDP antigen using manual epitype profiling. A new epitype profiling system was deployed by informatics vendor HLA Data Systems, developers of the mTilda Lab Management System and VxMatch, to ensure that DP alleles not covered by HLA antibody tests are incorporated into an electronic vXM workflow. Methods: The epitype profiling system contains a data table of the highly polymorphic DP hypervariable regions (HVR) for DPB1 alleles. There are a total of six HVR regions (HVR-A, B, C, D, E, F) containing the unique amino acid profile per allele and 291 epitype groups determined so far which correspond to alleles belonging to the same HVR motif. The system returns the HVR motifs for donor and alleles of the recipient antibodies and then sorts alleles of recipient antibodies by increasing reactivity. Total negative and positive HVR matches are based on a configurable positivity cutoff value. Matches are color-coded for investigation of possible DSA when there is a positive match that is accompanied by zero negative matches. If a DP antigen bead is negative, presence of antibodies to polymorphic motifs carried by the DP antigen can be excluded. Results: The computerized epitype profiling system was validated against our manual process for donors with alleles not contained within the assay, DPB1*16:01 and DPB1*85:01, and HVR motif profiling performed without error. With the increasing discovery of DP alleles, the system can cross-reference G-groups to determine HVR motifs for new alleles and can be configured to profile other HLA genes. Conclusions: Our evaluation suggests that extending vXM to include computerized epitype profiling is an efficient and reliable means to evaluate DP alleles not covered by antibody assays.
P153
COULD PBLS PROVIDE WITH ENOUGH B CELLS FOR A SUCCESSFUL FLOW CROSSMATCH? Runying Tian, Yan Li, Candy Young, Hua Zhu, Wendy Hanshew, Ping Rao, Dongfeng Chen. Duke Univeristy Medical Center, Durham, NC, United States. Aim: Although lymph nodes are preferred resources for isolation of lymphocytes for crossmatches, the nodes may not be available at the time of crossmatching. An unusual case led us to revisit 39 deceased donor’s crossmatch results to see if enough B cell events were acquired in flow crossmatching. Methods: Cells were isolated using Density Gradient of Ficoll-Hypaque from pronase-treated PBL, and/or from lymph nodes. Flow crossmatches (FCXM) were performed on a BD FACSCalibur. In FCXM, anti-CD3 was used to mark T-cells, anti-CD19 was used to mark B-cells and goat anti-human IgG was used as a secondary antibody for detection of donor specific antibody. Results: A 38 years old Caucasian female suffered a severe injury from a car accident was evaluated as the potential deceased donor for three kidney recipients. Three tubes of PBL were received for initial FCXM. The crossmatch failed due to lower B cell events (<400) acquired. Crossmatches were repeated using cells of lymph nodes. All crossmatches were negative and one of the three potential recipients was transplanted. We further analyzed 39 deceased donors. Out of the 39 donors, 23 had nodes at the time of crossmatch, 10 only had PBL, and 6 donors had both PBLs and nodes available. We found that T and B cell ratio was between 1:1 and 32:1 in PBL. 90% of donors fall in the range of 1:1 to 10:1. We further investigated the cause of donor death, WBC counts and medical histories but found no association between these factors with the T/B cell ratio or total lymphocyte number. All crossmatches with cells of lymph nodes were successful. Out of 16 crossmatches with cells of PBLs, only one (6%) did not have enough B cells. Conclusions: Our findings suggest the PBLs, as the resources of cells for crossmatch, most of the times (94%) could result in successful crossmatches.
P152
Abstracts / Human Immunology 79 (2018) 58–187
EVALUTION OF ELECTRONIC DP EPITYPE PROFILING TO DETERMINE DP DONOR-SPECIFIC ANTIBODIES DURING VIRTUAL CROSSMATCH Dong-Feng Chen 1, Wendy E. Hanshew 1, Divyank Saini 2, Barbara Ure 2, Collin Brack 2. 1 Duke University Medical Center, Durham, NC, United States; 2 HLA Data Systems, Houston, TX, United States. Aim: Our aim is to evaluate the utility of incorporating epitype analysis into the computerized virtual crossmatch (vXM) workflow for donors with DP alleles that are not covered by a recipient’s HLA antibody screening and identification assay. Our lab has used vXM to select deceased donors for transplantations for more than ten years. If the donor DP antigens are covered by the assay, the DSA can be determined directly by vXM. If the donor antigen is not included in the assay but is covered by an epitype (EDP) group which includes a DP antigen covered by the assay, the DSA can be determined based on the positivity of the EDP antigen using manual epitype profiling. A new epitype profiling system was deployed by informatics vendor HLA Data Systems, developers of the mTilda Lab Management System and VxMatch, to ensure that DP alleles not covered by HLA antibody tests are incorporated into an electronic vXM workflow. Methods: The epitype profiling system contains a data table of the highly polymorphic DP hypervariable regions (HVR) for DPB1 alleles. There are a total of six HVR regions (HVR-A, B, C, D, E, F) containing the unique amino acid profile per allele and 291 epitype groups determined so far which correspond to alleles belonging to the same HVR motif. The system returns the HVR motifs for donor and alleles of the recipient antibodies and then sorts alleles of recipient antibodies by increasing reactivity. Total negative and positive HVR matches are based on a configurable positivity cutoff value. Matches are color-coded for investigation of possible DSA when there is a positive match that is accompanied by zero negative matches. If a DP antigen bead is negative, presence of antibodies to polymorphic motifs carried by the DP antigen can be excluded. Results: The computerized epitype profiling system was validated against our manual process for donors with alleles not contained within the assay, DPB1*16:01 and DPB1*85:01, and HVR motif profiling performed without error. With the increasing discovery of DP alleles, the system can cross-reference G-groups to determine HVR motifs for new alleles and can be configured to profile other HLA genes. Conclusions: Our evaluation suggests that extending vXM to include computerized epitype profiling is an efficient and reliable means to evaluate DP alleles not covered by antibody assays.
P153
COULD PBLS PROVIDE WITH ENOUGH B CELLS FOR A SUCCESSFUL FLOW CROSSMATCH? Runying Tian, Yan Li, Candy Young, Hua Zhu, Wendy Hanshew, Ping Rao, Dongfeng Chen. Duke Univeristy Medical Center, Durham, NC, United States. Aim: Although lymph nodes are preferred resources for isolation of lymphocytes for crossmatches, the nodes may not be available at the time of crossmatching. An unusual case led us to revisit 39 deceased donor’s crossmatch results to see if enough B cell events were acquired in flow crossmatching. Methods: Cells were isolated using Density Gradient of Ficoll-Hypaque from pronase-treated PBL, and/or from lymph nodes. Flow crossmatches (FCXM) were performed on a BD FACSCalibur. In FCXM, anti-CD3 was used to mark T-cells, anti-CD19 was used to mark B-cells and goat anti-human IgG was used as a secondary antibody for detection of donor specific antibody. Results: A 38 years old Caucasian female suffered a severe injury from a car accident was evaluated as the potential deceased donor for three kidney recipients. Three tubes of PBL were received for initial FCXM. The crossmatch failed due to lower B cell events (<400) acquired. Crossmatches were repeated using cells of lymph nodes. All crossmatches were negative and one of the three potential recipients was transplanted. We further analyzed 39 deceased donors. Out of the 39 donors, 23 had nodes at the time of crossmatch, 10 only had PBL, and 6 donors had both PBLs and nodes available. We found that T and B cell ratio was between 1:1 and 32:1 in PBL. 90% of donors fall in the range of 1:1 to 10:1. We further investigated the cause of donor death, WBC counts and medical histories but found no association between these factors with the T/B cell ratio or total lymphocyte number. All crossmatches with cells of lymph nodes were successful. Out of 16 crossmatches with cells of PBLs, only one (6%) did not have enough B cells. Conclusions: Our findings suggest the PBLs, as the resources of cells for crossmatch, most of the times (94%) could result in successful crossmatches.