Evaluation of Lymphoproliferative Responses by Carboxy Fluorescein Succinimidyl Ester Assay in Heart Recipients With Infections L. Valor, E. Sarmiento, J. Navarro, A. Gallego, J. Fernandez-Yañez, E. Fernandez-Cruz, and J. Carbone ABSTRACT The analysis of proliferative responses using 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in flow cytometry is widely used to assess lymphocyte function. The aim of this study was to evaluate nonspecific and specific lymphoproliferative responses using CFSE in heart recipients before and after transplantation and their association with the development of infection. We used four-color flow cytometry to measure the response of peripheral CD3+, CD4+, and CD8+ T cells to phytohemagglutinin mitogen (PHA), tetanus toxoid, hepatitis B, and influenza vaccines using a CFSE proliferation assay in 12 heart recipients and 8 healthy control subjects. Recipients were prospectively evaluated. Immunological studies were performed before and at 3 months after transplantation. A 12-month clinical follow-up examination sought to detect the prevalence of severe infectious complications. Heart recipients (infected [n = 7] and uninfected [n = 5]) disclosed significantly lower percentages of proliferative responses than healthy controls against PHA at both study points. Baseline CD3+, CD4+, and CD8+, antitetanus proliferative responses were significantly lower in infected heart recipients than controls. Patients who developed infections displayed significantly lower percentages of CD3+CFSE and CD8+CFSE cells to PHA mitogen at 3 months after transplantation versus those without infections. In conclusion, nonspecific T-cell reactivity to PHA was lower in heart recipients with posttransplantation infections. I NFECTION is an important cause of morbidity and mortality after heart transplantation. 1,2 Identification of risk factors would help to reduce morbidity and mortality allowing implementation of preventive measures for high- risk patients. The fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) labels intracellular proteins. The fluorescence of each labeled cell is divided equally between daughter cells, thus enabling flow cytom- etry to determine the number of cells produced in each generation. 3 Although equivalent to the classic 3H-thymidine incorporation assay, the CFSE assay has significant advan- tages in terms of the ability to gate specific populations of lymphocytes. 4,5 In the present study, we used the CFSE assay to evaluate mitogen- and vaccine-specific responses among patients undergoing heart transplantation and their associations with infection. METHODS This case controlled study prospectively evaluated lymphoprolif- erative responses in 12 heart recipients of mean age 56 years [range, 33 to 67 years]) who received prophylactic immunizations at the time of inclusion on the waiting list. Patients who were retransplanted were not included. We included 8 age-matched healthy individuals (of mean age 54 years [range, 40 to 62 years]) as controls. The same immunologists and cardiologists reviewed the patient records over 12 months after transplantation. All patients received induction therapy with two doses of the interleukin-2 receptor antagonist daclizumab. Maintenance immunosuppression included mycophenolate mofetil, prednisone, and tacrolimus. Before enter- ing the heart transplantation protocol, all patients were immunized against pneumococcus (1 dose of 23-serotypes antipneumococcal vaccine), influenza (1 dose), tetanus toxoid (1 dose), and hepatitis B (HB, 3 doses). Antimicrobial prophylaxis included cephazolin (2 From the Clinical Immunology Department, Gregorio Marañon University Hospital, Madrid, Spain. Supported by the Instituto de Salud Carlos III, projects FIS 05/0839, FIS 08/1430. Address reprint requests to Javier Carbone, Clinical Immunol- ogy Department, Gregorio Marañon University Hospital, Dr. Esquerdo, 46, 28007, Madrid, Spain. E-mail: jcarbone.hgugm@ salud.madrid.org © 2012 by Elsevier Inc. All rights reserved. 0041-1345/–see front matter 360 Park Avenue South, New York, NY 10010-1710 http://dx.doi.org/10.1016/j.transproceed.2012.09.054 Transplantation Proceedings, 44, 2649 –2652 (2012) 2649