Ex vivo evidence of lymphocyte apoptosis in hairy cell leukemia, induced by 2-chlorodeoxyadenosine treatment

In all living cells phosphatidylserine (PS) is located at the cytosol side of the membrane and becomes exposed at the cell surface only during necrosis or apoptosis. This phenomenon allows measurement of cell death on a cell-by-cell basis, using labeled Annexin V, which has a strong affinity to PS. Two patients with hairy cell leukemia (HCL) who had relapsed after splenectomy and alpha-interferon therapy were treated with 2-chlorodeoxyadenosine (2-CdA) for 7 days. Blood samples were taken from the start of therapy until day 22. Percentages of HCL cells, T cells, B cells, and NK cells were measured with PE-labeled monoclonal antibodies by flow cytometry (FCM). The absolute lymphocyte count dropped rapidly to almost zero in both patients within 7 days. The disappearance rate of lymphocyte subfractions did not show a specific pattern. The percentage of apoptosis in lymphocyte subfractions was measured in freshly prepared cell samples by FCM with FITC-labeled Annexin V in the propidium iodide-negative (non-necrotic) cell fraction. Percentages of PS-positive cells increased gradually till a nadir of Annexin V positivity was reached at 14 and 16 days. Because during the first week the absolute cell counts became almost zero, the absolute numbers of PS-positive cells were still extremely low, i.e., less than 0.1x10(9)/l. Nevertheless, we observed apoptotic cells in circulation after 2-CdA therapy. To our knowledge, this is the first report of the occurrence of apoptosis ex vivo in circulating blood cells after cytotoxic therapy.