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.

Ethylenediaminetetraacetic acid plus citrate-theophylline-adenosine-dipyridamole (EDTA-CTAD): a novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood.

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant recommended for full blood counts, citrate is recommended for coagulation and platelet studies, and citrate-theophylline-adenosine-dipyridamole (CTAD) inhibits platelet activation. Because the combination of EDTA and CTAD (E/C) is better than EDTA or CTAD alone for measuring platelet parameters on the ADVIA 120 Haematology System, we investigated whether it also offers advantages for the flow cytometric assessment of platelet and/or neutrophil activation and platelet-leucocyte aggregate formation ex vivo. Blood from healthy subjects was collected into E/C or citrate, kept at room temperature or at 4 degrees C, and analysed 0 to 360 min later in the ADVIA 120 and by immunofluorescent flow cytometry. Platelet count, mean platelet volume, number of platelet clumps, mean platelet component, numbers of CD62P(+) platelets and platelet-leucocyte aggregates, and expression of CD11b on neutrophils changed little over 360 min in blood with E/C kept at 4 degrees C. In contrast, one or more parameter changed when blood was kept with E/C at ambient temperature or with citrate at either temperature. The use of E/C in in vitro and in vivo studies is illustrated. Platelet and neutrophil activation status ex vivo can be reliably assessed if blood is collected into E/C, held at 4 degrees C, and analysed within 6 h.

Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120 hematology system.

BACKGROUND: Monitoring of platelet activation by the ADVIA 120 Hematology System requires an anticoagulant and protocol that ensures that platelets are sphered and their activation status is not altered artifactually in vitro. METHODS: Blood from healthy controls was collected into tripotassium EDTA; citrate, theophylline, adenosine, and dipyridamole (CTAD); or a combination of both (E/C) and stored at ambient temperature or at 4 degrees C (E/C only) and then analyzed between 0 and 180 min later on the ADVIA 120. In addition, immunofluorescent flow cytometry was used to identify activated platelets and platelet-leukocyte aggregates. RESULTS: In blood stored with all three anticoagulants, the platelet count changed little, but the mean platelet volume (MPV) at first decreased and then increased, whereas the mean platelet component (MPC; an indicator of activation) changed in a reciprocal manner. The changes in MPV and MPC, which reflect platelet sphering and swelling, were greatest between 30 and 60 min in blood stored at ambient temperature, irrespective of which anticoagulant was used, and between 60 and 180 min when blood anticoagulated with E/C was stored at 4 degrees C. In all anticoagulants, the percentages of platelets expressing CD62P and of leukocytes in platelet-leukocyte aggregates increased significantly (P <0.01) over 180 min at ambient temperature. Only minimal (<2%) increases occurred when blood with E/C was stored at 4 degrees C. CONCLUSIONS: When determining platelet activation ex vivo on the ADVIA 120, blood should be collected into E/C, stored at 4 degrees C, and analyzed between 60 and 180 min later; these conditions ensure maximum platelet sphering without concurrent artifactual platelet activation.