Flow cytometric findings in platelets of patients following allogeneic hematopoietic stem cell transplantation

Bone Marrow Transplantation (2002) 30, 381–387  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt Platelet measurements Flow cytometric findings in platelets of patients following allogeneic hematopoietic stem cell transplantation R Pihusch1, H Wegner1, C Salat1, M Pihusch1, E Holler2, HJ Kolb1,3 and E Hiller1 1 Medizinische Klinik III – Groβhadern, Klinikum der Ludwig Maximilians-Universität München, Germany; 2Abteilung für Hämatologie und Internistische Onkologie, Klinikum der Universität Regensburg, Germany; and 3Klinische Kooperationsgruppe Hämatopoietische Zelltransplantation, GSF, Forschungszentrum für Umwelt und Gesundheit, Neuherberg, Germany Summary: Following allogeneic hematopoietic stem cell transplantation (HSCT) patients may have an increased bleeding tendency in spite of a normal platelet count. Moreover, an association between chronic graft-versus-host disease (cGVHD) and a thrombophilic state has been observed. Platelet receptors and granules from 27 patients following HSCT (13 without cGVHD, 14 with cGVHD) were evaluated by flow cytometric analysis and compared to 62 healthy controls. Platelets from HSCT patients stained weakly with mepacrine indicating a reduced content of dense bodies, whereas no significant degranulation reaction of alpha granules and lysosomes was detectable. In addition, a lower surface expression of GP Ia/IIa was observed, indicating an acquired thrombocytopathy. The surface receptors are activated in HSCT patients, which could be seen by the lower surface expression of GP Ib internalized during the activation process and elevated levels of LIBS-1 and PAC-1 antibody binding. Patients with cGVHD had a seven-fold increased ratio of microparticles. This study demonstrates platelet receptor and granule defects in patients following HSCT. The key role of platelets in HSCTassociated hemostatic disorders is underscored by the high levels of circulating microparticles in cGvHD patients which might explain the thrombophilic state in these patients. Bone Marrow Transplantation (2002) 30, 381–387. doi:10.1038/sj.bmt.1703663 Keywords: platelets; graft-versus-host reaction; storage pool disease; microparticles Platelet function is strongly dependent on glycoproteins (GPs) exposed on the surface of activated platelets.1–3 The GP IIb/IIIa complex is the inducible fibrinogen receptor on platelets and plays a central role in platelet aggregation. This receptor has the capacity to shift through conforCorrespondence: Dr med R Pihusch, Medizinische Klinik III – Groβhadern, Klinikum der Ludwig Maximilians-Universität München, Marchioninistr. 15, 81377 München, Germany Received 18 February 2002; accepted 29 May 2002 mational changes from a low-affinity state to a ligand-competent state on activated platelets, which can be detected by PAC-1-antibody. Furthermore, fibrinogen binding to GP IIb/IIIa itself modifies the conformation of this integrin, exposing neoepitopes known as ligand-induced binding sites (LIBS). The GP Ib/V/IX complex is the von Willebrand factor (vWF) receptor responsible for adhesion of platelets to a damaged vessel wall, whereas the glycoprotein complex Ia/IIa (GP Ia/IIa) is a major collagen receptor on platelets. The latter plays an important role in platelet adhesion and activation following initial interaction of the platelet with the vessel wall which is mediated by vWF.4 During aggregation platelets degranulate and express multiple granule-stored GPs on their surface that mediate platelet interactions with other vascular cells including leukocytes and the endothelium. Following intensive activation, platelets liberate minute membrane vesicles. These microparticles can be detected in the circulation and induce a significant thrombosis risk by providing a large phospholipid surface which serves as substrate for plasmatic coagulation.5 A common clinical observation following hematopoietic stem cell transplantation (HSCT) is an impaired bleeding time in spite of a normal platelet count. On the other hand, patients with chronic graft-versus-host disease (cGVHD) have a 13-fold thrombosis risk in spite of thrombocytopenia.6 In this study, we evaluated changes in membrane GPs on circulating platelets and the liberation of microparticles in long-term survivors of allogeneic hematopoietic stem cell transplantation and found profound changes which could well explain the clinical observations. Patients and methods Patients A random series of 27 patients which had undergone allogeneic hematopoietic stem cell (HSCT) transplantation months ago were evaluated. Patients were regularly monitored on an outpatient basis. Fourteen patients showed signs of chronic GVHD (skin, liver, gut). Of the remaining 13 patients having no evidence of cGVHD, six patients were thrombocytopenic due to a delayed platelet take. All Stem cell transplantation and platelets R Pihusch et al 382 patients were in complete remission from their underlying disease and had a complete chimerism. None of the patients had any bacterial, fungal or viral infections within 2 months prior to the beginning of the study, nor did any have implanted stents or a history of antirheumatic medication. Platelet transfusions had not been given within the last 10 days. In addition, a control group of 62 healthy individuals recruited from the medical staff was evaluated. The patient and transplant characteristics are given in Table 1. stopped by adding 2 ml paraformaldehyde (2% solution, pH 7.4) and the probe was scanned immediately in a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany) equipped with a 15 mW argon ion laser. By electronic gating (see Figure 1), 30 000 CD41-positive single platelets were acquired from each sample and analyzed using CellQuest software (Becton Dickinson). Specific antibodies and in vitro stimulation Fluorescein-tagged antibodies against the following antigens were used (all Immunotech, Hamburg, Germany): Laboratory parameters Laboratory investigations included a routine platelet count (H 6000 analyser, Coulter, Miami, FL, USA) and platelet flow cytometry. Platelet flow cytometry is an in vitro technique which allows analysis of surface epitopes on platelets with specific fluorochrome tagged antibodies.7 The method used was based on that of the European Working Group on Clinical Cell Analysis8 with additional in vitro stimulation of platelets to gain information on the platelet function. 104 Table 1 R2 CD 41-PE 102 R4 R5 1 10 100 100 101 102 FSC - Height Demographic and transplantation characteristics of the patients under study and controls Total Gender (male/female) Age (median years (range)) Diagnosis Chronic myelogenous leukemia Acute myelogenous leukemia Acute lymphatic leukemia Non-Hodgkin’s lymphoma Myelodysplastic syndrome Osteomyelofibrosis Type of allogeneic transplantation Related Unrelated HLA identical Unrelated HLA different Time after HSCT (median weeks ( range)) (...truncated)