SHORT REPORT Increased apoptotic cells in bone marrow biopsies from patients with aplastic anaemia F ERNANDO C ALLERA AND ROBERTO P. FALCA ˜ O Department of Clinical Medicine, School of Medicine, Ribeira ˜o Preto, Sa ˜o Paulo, Brazil Received 14 November 1996; accepted for publication 2 April 1997 Summary. In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anaemia (AA) we determined the proportion of apoptotic cells in paraffin- embedded bone marrow biopsies from patients with aplastic anaemia using an in situ TdT-catalysed DNA nick end labelling (TUNEL) staining method. A significant increase in the proportion of mononuclear apoptotic cells was demon- strated in biopsies from patients with aplastic anaemia (8 . 19  1 . 45%) when compared with controls (2 . 07  0 . 86%). These data support the view that apoptosis may play a role in the pathophysiology of bone marrow failure. Keywords: aplastic anaemia, apoptosis, bone marrow biopsy, stem cells, TUNEL method. Apoptosis is a morphologically distinct form of programmed cell death that plays a critical role in the homeostasis of haemopoietic stem cells controlling the rate of production of committed cells through the sensitivity of progenitor cells to the presence or absence of survival factors (Kerr et al, 1972; Williams et al, 1990). Recent evidence suggests that alterations in apoptosis contribute to the pathogenesis of human diseases including cancer, viral infections and autoimmune diseases (Thompson, 1995). Furthermore, other studies have supported the view that apoptosis is involved in the pathophysiology of bone marrow failure in aplastic anaemia (AA). Maciejewski et al (1995) have shown that Fas-receptor (Itoh et al, 1991) expression is increased in bone marrow CD34 þ cells of patients with AA. In addition, we have demonstrated that Fas receptor is also over- expressed in bone marrow and peripheral blood lymphocytes of patients with aplastic anaemia (Callera et al, 1995). Finally, Philpott et al (1995) have shown that a significantly larger proportion of CD34 þ progenitor cells from aplastic anaemia bone marrow were apoptotic as compared with normal CD34 þ bone marrow when estimated by flow cytometry; the most marked difference was observed in the more severely affected AA patients. However, to our knowl- edge, there are no data concerning the in situ detection of apoptotic cells in bone marrow biopsies of patients with aplastic anaemia. Therefore we investigated the proportion of mononuclear apoptotic cells in AA marrow biopsies by using an in situ TdT-catalysed DNA nick end labelling (TUNEL) staining method (Gavrieli et al, 1992). MATERIAL AND METHODS Posterior iliac crest bone marrow biopsies were obtained from 11 patients with AA aged 16–65 years (five females and six males). Four had severe and seven moderate aplastic anaemia (Camitta et al, 1976). The values (median, range) of haemoglobin, neutrophil and platelet counts were 5 . 8 g/dl (1 . 9–10 . 9), 0 . 8 × 10 9 /l (1 . 4–2 . 4) and 9 × 10 9 /l (2–20) respectively. All patients were transfusion dependent. Five patients had not been previously treated and six were patients who had relapsed after immunosuppressive therapy or who did not respond to therapy. Marrow biopsies without tumour involvement or histological alterations obtained from 12 patients with stage I Hodgkin disease were studied as controls. Bouin-fixed bone marrow biopsies were embedded in paraffin and 4–6 mm paraffin sections were adhered to slides followed by decalcification. Apoptotic cells were assessed in the deparaffinized and rehydrated slides using in situ terminal deoxynucleotidyl transferase- catalysed DNA nick end labelling (TUNEL) staining (ApopTag TM plus peroxidase kit, ONCOR Inc., U.S.A.). The percentage of positive cells (peroxidase-stained nuclei) was determined by counting 500 mononuclear cells from each slide. British Journal of Haematology , 1997, 98, 18–20 18  1997 Blackwell Science Ltd Correspondence: Dr Roberto Passetto Falca ˜o, Department of Clinical Medicine, School of Medicine, Ribeira ˜o Preto, Sa ˜o Paulo, Av. Bandeirantes 3900, 14049-900, Brazil.