0041-1337/01/7103-422/0 TRANSPLANTATION Vol. 71, 422–428, No. 3, February 15, 2001 Copyright © 2001 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. IMPROVED FLOW CYTOMETRIC DETECTION OF HLA ALLOANTIBODIES USING PRONASE POTENTIAL IMPLICATIONS IN RENAL TRANSPLANTATION SMITA VAIDYA, 1,2 TODD Y. COOPER, 1 JEANNE AVANDSALEHI, 1 TITUS BARNES, 1 KARL BROOKS, 1 PHOUMYMALA HYMEL, 1 MARYAM NOOR, 1 RACHEL SELLERS, 1 ALICE THOMAS, 1 DOD STEWART, 3 JOHN DALLER, 4 JAY C. FISH, 4 KRISTENE K. GUGLIUZZA, 4 AND ROBERT A. BRAY 5 Departments of Pathology and Surgery, University of Texas Medical Branch, Galveston, Texas; Ochsner Clinic, New Orleans, Louisiana; and Department of Pathology, Emory University Hospital, Atlanta, Georgia Background. Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the “standard of practice” in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-re- ceptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. Methods. Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pro- nase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. Results. After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 7810 to 574(P<0.05) and 10711 to 493 (P<0.00001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-pos- itive reaction observed. In this study, pronase treat- ment improved the specificity of B cell FCXM for de- tecting class II antibodies from 75% to 100% (P0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evalu- ated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identi- fied in each case. Conclusion. Utilization of pronase-treated lympho- cytes improves both the sensitivity and specificity of the FCXM. INTRODUCTION The UNOS 1999 Annual Report suggests that, despite significant improvement and progress in immunosuppressive therapy, approximately 8 –10% of all transplanted kidneys (i.e., more than1000 kidney per year) are lost within the first 3 months (1). A significant portion of this graft loss is directly attributable to immunological rejection (1). Numerous stud- ies, including our own (2, 5), have shown that preformed HLA antibodies continue to play a major role in early allograft loss. These antibodies are low titer and generally undetect- able even in the most sensitive complement-mediated cyto- toxicity (CDC) assays. However, these antibodies are readily detectable by the flow cytometry crossmatch (FCXM) (4–6). For this reason, the FCXM has become the “standard of practice” for many transplant centers (7–10). Interestingly, since its inception in 1983 by Garovoy et al. (11) and its adaption to a multicolor method by Bray et al.(12), the actual FCXM method has remained essentially unchanged. Al- though interpretation of the T cell FCXM is straightforward and uncomplicated, the B cell FCXM has remained problem- atic owing to the presence of Fc receptors on the cell surface (13). These Fc receptors are also found on the T-cell surface, albeit to a lesser proportion than on the B-cell surface (14). Normal or irrelevant IgG can bind to such T and B cells, especially when sera contain IgG in an aggregated or com- plexed form (15–16). Consequently, an increase in fluores- cence may be interpreted as a positive result when, in fact, it may only indicate the presence of excess irrelevant IgG bound to Fc receptors. To improve the specificity and sensitivity of FCXM, we have used the proteolytic enzyme pronase to remove Fc re- ceptors from the surfaces of T and B cells before their use in FCXM. In this report, we demonstrate that pronase treat- ment significantly decreases the nonspecific background flu- orescence, thereby enhancing the signal-to-noise separation and consequently increasing the sensitivity and specificity of 1 Department of Pathology, University of Texas Medical Branch. 2 Address correspondence to: Smita Vaidya, PhD, The University of Texas Medical Branch, Tissue Antigen Laboratory, Rebecca Sealy Hospital, Suite 2.810, 301 University Boulevard, Galveston, TX 77555-0178. 3 Ochsner Clinic. 4 Department of Surgery, University of Texas Medical Branch. 5 Department of Pathology, Emory University Hospital. 422