High Levels of Circulating Monocyte-Platelet Aggregates Can Predict Rejection Episodes After Orthotopic Liver Trasplantation R. Vanacore, C. Guida, P. Urciuoli, A. Mazzoni, I. Bianco, L. Urbani, G. Stampacchia, F. Filipponi, and F. Scatena T HE RELEVANCE of the vascular endothelium to the pathological damage that follows graft rejection is well known; cell-associated adhesion molecules are consid- ered early markers of bioincompatibility. 1 Endothelium is the interface between the donor organ and the recipient immune system. A well-known consequence of the endo- thelial damage is platelet activation, a clinical situation that may be evaluated by studying the expression of activation markers, such as CD62p or Annexin V, and recently with flow cytometric analysis circulating monocyte-platelet ag- gregates. 2,3 These mechanisms may also play an important role in liver transplantation. METHODS We evaluated 25 liver transplant patients of whom 15 were in good clinical condition and 10 in an initial rejection phase, including 8 tested also after clinical resolution. Aliquots (100 L) of peripheral blood, collected in EDTA Vacutainers, were incubated for 20 minutes with 20 L of phycoerythrin (PE). Conjugated anti-CD14 to reveal monocytes and 20 L of fluorescin isothiocyanate (FITC) conjugated anti-CD61 FITC to reveal platelets (monoclonal anti- bodies were provided by BD Bioscience). After the red blood cells were lysed using NH 4 Cl the flow cytometric analysis was performed using a FacsCalibur instrument (BD Bioscience). The Cell Quest software permitted calculation of the percentage of monocytes with adhesed platelets. RESULTS Our results showed an increased percentage of monocytes with platelet aggregates among patients in rejection phase (39%  19.1%) compared with those without symptoms (15.6%  9.3%)(P  .0001). The eight subjects tested after remission of clinical symptoms showed results that were similar to those of patients who did not display rejection (16.5%  7.1%). CONCLUSIONS Platelets may become activated in a number of clinical disorders, participating in thrombus formation. We sought to develop a direct test for activated platelets in whole blood using flow cytometry. Recent observations indicate that polymorphonuclear leukocyte adhesion to activated platelets is important for the recruitment of these cells to sites of vascular damage and thrombus formation. 4 More- over, another study showed a correlation between platelet and leukocyte activation and the severity of septic organ dysfunction. 5 Our preliminary results indicate that the formation of monocyte-platelet aggregates may represent a useful marker of liver rejection risk. This approach is rapid and not expensive; it may be used for follow-up assessment in association with other clinical parameters in order to re- duce the utilization of more invasive, dangerous, and ex- pensive techniques such as liver biopsy. REFERENCES 1. Carreno MP, Stuard S, Bonomini M, et al: Nephrol Dial Transplant 11:2248, 1996 2. Powell CC, Rohrer MJ, Barnard MR, et al: J Vasc Surg 30:844, 1999 3. Michelson AD, Barnard MR, Krueger LA, et al: Circulation 104:1533, 2001 4. Evangelista V, Manarini S, Sideri R, et al: Blood 93:876, 1999 5. Russwurm S, Vickers J, Meier-Hellmann A, et al: Shock 17:263, 2002 From the Blood Bank and Liver Transplant Unit, Cisanello Hospital, Pisa, Italy. Address reprint requests to Dr R. Vanacore, Immunoemalogy II, Cisanello Hospital, Via Paradisa 2, 56100 Pisa, Italy. E-mail: r.vanacore@mail.ao-pisa.toscana.it © 2003 by Elsevier Science Inc. 0041-1345/03/$–see front matter 360 Park Avenue South, New York, NY 10010-1710 doi:10.1016/S0041-1345(03)00252-5 Transplantation Proceedings, 35, 1019 (2003) 1019