A Novel DSA Flow Cytometer Crossmatch (DSA-FXM) to Eliminate Ambiguous Auto- and Allo-FXM Results
Pathology, Stanford University, Palo Alto, CA.
Meeting: 2015 American Transplant Congress
Abstract number: A88
Keywords: Antibodies, Autoimmunity, FACS analysis, Kidney transplantation
Session Information
Session Name: Poster Session A: Kidney Antibody Mediated Rejection
Session Type: Poster Session
Date: Saturday, May 2, 2015
Session Time: 5:30pm-7:30pm
Presentation Time: 5:30pm-7:30pm
Location: Exhibit Hall E
Introduction:
Though usually rejected on theoretical grounds, a positive Auto-FXM could result from either or both auto-reactive non-HLA or HLA-antibodies (Ab). Furthermore, a positive Auto-FXM serum very often causes a positive Allo-FXM and increases the difficulty in interpreting the final FXM results. There are no existing methods available to objectively interpret those ambiguous FXM results. A novel DSA-FXM was recently developed in our lab to selectively detect only class I (CI) and II (CII) HLA-Abs bound to native HLA expressed on the target cell surface. It was able to distinguish HLA-Ab from Non-HLA-Ab, Auto-DSA from Allo-DSA, eliminate false positive Allo-FXM results caused by non-HLA-Abs, and, in the case of true positive Allo-FXM, determine whether the antibody detected was to CI or CII.
Methods:
DSA-FXM: 0.2X106 donor PBMCs were incubated with recipient serum at RT for 30'; after washes, beads conjugated with anti-HLA class-I or -II MoAbs were added simultaneously. The cells were lysed in lysis buffer containing PE-anti-IgG and the complexes of HLA-Ag/DSA/PE-anti-IgG were captured by the CI or CII beads. Beads were acquired on a flow cytometer. The PE intensities on CI and CII beads were proportionately correlated with the HLA-DSA levels in the serum.
FXM: General IgG FXM procedure. Luminex Single Antigen Beads (L-SAB): One Lambda method
Results:
A total of 68 positive Auto-FXM and 41 simultaneous Allo-FXMs were performed. 39/41 Allo-FXM (95%) were also positive. By DSA-FXM, 46 (68%) positive Auto-FXMs were caused by Non-HLA-Abs and 22 (32%) positive Auto-FXM were caused by Auto-DSA. L-SAB Auto-DSAs were detected in 3 (7%) of DSA-FXM negative group but 7 (32%) of DSA-FXM positive group.
Conclusions:
Auto-HLA-Ab (Auto-DSA) exists and can cause a positive Auto-FXM. Non-HLA-Ab can cause false positive Auto- and Allo-FXMs. The DSA-FXM method is able to selectively detect true DSA against native HLA-Ag on both recipient and donor cell surfaces and eliminate false positive or ambiguous FXM results. This allows accurate assessment of donor compatibility, in addition to providing information on the antibody class (I or II) contributing to the positive result.
To cite this abstract in AMA style:
Chen G, Lin L, Tyan D. A Novel DSA Flow Cytometer Crossmatch (DSA-FXM) to Eliminate Ambiguous Auto- and Allo-FXM Results [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/a-novel-dsa-flow-cytometer-crossmatch-dsa-fxm-to-eliminate-ambiguous-auto-and-allo-fxm-results/. Accessed May 17, 2025.« Back to 2015 American Transplant Congress