A simple, novel, procedure for monitoring membrane scrambling and permeability in microparticles, platelets, and leukocytes in whole blood samples.

OBJECTIVE: To devise and evaluate a protocol for monitoring lipid packing and membrane permeability in live cells in whole peripheral blood, and to assess these properties in blood from controls and patients. MATERIALS AND METHODS: Samples were stained simultaneously with merocyanine 540 and Sytox Green, diluted, and analyzed by three-color flow cytometry. RESULTS: Membrane changes characteristic of apoptosis/necrosis were detected in cells in culture and in blood that had been "aged" in vitro, with sensitivity and specificity, which was comparable to that obtained using fluoresceinated Annexin-V and ethidium bromide or propidium iodide. Merocyanine 540 also reported increases in membrane lipid disorder when cells in whole blood were activated by the Ca(2+) ionophore, A23187. Very few (<2%) leukocytes or platelets in the blood of healthy subjects (n = 14) had disordered and/or permeable membranes, However, if blood was stored with heparin the microparticle to platelet ratio increased and membrane lipids of microparticles and platelets became disordered within hours. In the blood of patients with idiopathic thrombocytopenia (n = 4), the microparticle to platelet ratio (1.5 +/- 1.1 vs 0.14 +/- 0.06; p = 0.000), and the percentages of microparticles (67.3% +/- 34.9% vs 20.4% +/- 12.6%; p = 0.000) and platelets (47.0% +/- 18.8% vs 1.9 +/- 2.0%; p = 0.000) with disordered membrane lipids were all markedly increased by comparison with the controls. CONCLUSIONS: This stain combination enabled important membrane characteristics to be assessed simply and quickly in unfixed, whole blood; and revealed significant differences in patient blood.

The neutropenia induced by the thalidomide analogue CC-4047 in patients with multiple myeloma is associated with an increased percentage of neutrophils bearing CD64.

A major limitation to the treatment of multiple myeloma by the thalidomide analogue CC-4047 (Actimid) is the development of a severe neutropenia. We investigated the hypothesis that this effect may have been due to CC-4047 enhancing the removal of neutrophils from the circulation by altering the expression of surface adhesion molecules required for endothelial binding, by binding to platelets, or by enhancing apoptosis. Flow cytometric analysis was used to examine the expression of neutrophil surface molecules, platelet binding and apoptosis in whole blood samples from 19 patients with multiple myeloma who were assigned to receive either 1, 2, 5 or 10 mg of CC-4047 every other day (e.o.d.) for 28 days. CC-4047 induced dose-related decreases in neutrophil numbers and increases in the percentage of CD64-positive neutrophils, but had little, or no effect on the expression of CD11b, CD62L or CD162, neutrophil-platelet binding, or apoptosis. Relative decreases in the neutrophil count were inversely associated with relative increases in the intensity of CD64 expression on neutrophils (r=- 0.307; p=0.028). Although seven patients developed severe neutropenia, none suffered severe or recurrent bacterial infections. The percentage of CD64-positive neutrophils was still increased in eight patients who continued receiving 1-5 mg CC-4047 e.o.d. for several months afterwards, but neutrophil counts were similar to pre-treatment values.