Flow Cytometric Measurement of ABO Antibodies in ABO-Incompatible Living Donor Kidney Transplantation Gisella Puga Yung, 1 Piero V. Valli, 1 Astrid Starke, 2 Regula J. Mueller, 1 Thomas Fehr, 2 Marija Cesar-O ¨ zpamir, 3 Urs Schanz, 3 Markus Weber, 4 Rudolf P. Wu ¨thrich, 2 Jo ¨rg D. Seebach, 1,5 and Georg Stussi 1 Due to different detection methods, a comparison of anti-A/B antibody (Ab) levels among transplantation centers after living donor ABO-incompatible kidney transplantation is problematic. In the present study, anti-A/B Ab levels were determined prior to, and after, blood group A-to-O kidney transplantation using a recently established semiquantita- tive flow cytometry-based method, ABO fluorescence-activated cell sorting (ABO-FACS), and compared with standard agglutination titers and indirect antiglobulin testing. Pretransplant agglutination titers were reduced from 1:64 to 1:4, by a total of 14 Glycosorb A column immunoadsorptions (IADSs). Compared with the agglutination titers, antidonor immunoglobulin (Ig) M ABO-FACS mean fluorescence intensity ratios (MFIRs) decreased faster and remained low. No difference was observed using donor type or third-party A red blood cells (RBCs) for the ABO-FACS. Glycosorb A columns were not specific, also reducing anti-B and antiporcine IgM levels, which was confirmed by detecting anti-A/B and antiporcine Abs in the column eluates. In conclusion, analysis of pre- and posttransplant Abs from ABO-incompatible kidney transplant recipients by ABO-FACS allows a better understanding of Ab kinetics, which may improve the design of future IADS protocols. Keywords: ABO fluorescence-activated cell sorting (ABO-FACS), ABO-incompatible living donor kidney transplantation. (Transplantation 2007;84: S20–S23) O ver the past few years, ABO-incompatible living donor kidney transplantation has become a valuable approach to enlarging the available donor pool (1–6). According to Landsteiner’s seminal discovery, individuals with blood group A produce anti-B antibodies (Ab), while individuals with blood group B produce anti-A Ab, and individuals with blood group O produce both anti-A and anti-B Ab. Since ABO antigen (Ag) is expressed not only on red blood cells (RBCs), but also on endothelial cells (ECs) (7, 8), anti-A/B Ab can bind to ECs of the donor organ and induce (hyper-) acute humoral graft rejection. Therefore, circulating anti-A/B Abs need to be reduced in the recipient’s serum prior to transplanta- tion in order to overcome anti-A/B Ab-mediated rejection and early graft loss in ABO-incompatible kidney transplantation (5, 9, 10). To achieve this goal, several techniques have been reported, including Ag-specific immunoadsorption (IADS), plasma exchange, and double filtration (1, 4, 5, 11). Similarly to the ABO Ag, the carbohydrate Gal1-3Gal (Gal) Ag, which is ubiquitously expressed in vertebrates except in hu- mans and anthropoid apes, stimulates the generation of anti- Gal Ab in humans (8, 12). These anti-Gal Abs bind to porcine cells, causing hyperacute rejection in xenotransplan- tation (13), and represent an excellent positive control to evaluate anti-A/B Ab. Standard immunohematologic evaluation of anti-A/B Ab titers includes blood group typing, agglutination, and in- direct antiglobulin testing (IAT), providing only indirect ev- idence for the presence of immunoglobulin (Ig) G Abs and being unable to detect IgG subclasses. The major disadvan- tage of these assays is the lack of standardized protocols, with considerable interobserver and interassay variability, as well as the failure to clearly discriminate IgG from IgM Abs. The former leads to a poor comparability of the titers originating from different centers. The latter contributes to the confusion about the role of IgG (subclasses) versus IgM Abs in humoral rejection. To resolve these problems, we utilized a recently established semiquantitative flow cytometry-based method, ABO fluorescence-activated cell sorting (ABO-FACS), to measure anti-A/B and antiporcine IgM/IgG Ab serum levels prior to, and after, a living-donor ABO-incompatible kidney transplantation (14, 15). MATERIALS AND METHODS ABO Fluorescence-Activated Cell Sorting The ABO-FACS method has been described in detail elsewhere (14). In brief, rhesus-negative A or B RBCs, donor RBCs, and porcine RBCs were fixed with formaldehyde/glu- taraldehyde to avoid agglutination, and were stored for sev- The authors declare no potential conflicts of interest. G. Puga Yung and G. Stussi contributed equally to this manuscript. Supported by grants from the Swiss National Science Foundation (4046- 58668), the University of Zurich (FK 54170101), Union Banque Swiss AG on behalf of a client, and the Krebsliga Zu ¨rich. 1 Laboratory for Transplantation Immunology, University Hospital Zurich, Zurich, Switzerland. 2 Clinic of Nephrology, University Hospital Zurich, Zurich, Switzerland. 3 Clinic of Hematology, Department of Internal Medicine, University Hos- pital Zurich, Zurich, Switzerland. 4 Department of Visceral and Transplant Surgery, University Hospital Zu- rich, Zurich, Switzerland. 5 Address correspondence to: Jo ¨rg D. Seebach, M.D., University Hospital Zu- rich, Department of Internal Medicine, Laboratory for Transplantation Im- munology, Ra ¨mistrasse 100, C HOER 31, CH-8091 Zurich, Switzerland. E-mail: klinseeb@usz.unizh.ch Copyright © 2007 by Lippincott Williams & Wilkins ISSN 0041-1337/07/84012S-20 DOI: 10.1097/01.tp.0000296646.17845.12 S20 Transplantation • Volume 84, Number 12S, December 27, 2007