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.


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.
McCarthy
Desmond A
DA
School of Biological and Chemical Sciences, Queen Mary (University of London), London, UK. d.mccarthy@qmul.ac.uk
Macey
Marion G
MG
Allen
Paul D
PD
D000328:Adult; D000368:Aged; D017209:Apoptosis; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D008875:Middle Aged; D009930:Organic Chemicals / Q000493:pharmacokinetics; D011744:Pyrimidinones / Q000493:pharmacokinetics; D012016:Reference Values; D013194:Staining and Labeling / Q000379:methods*; D013921:Thrombocytopenia / Q000097:blood*
D005456:Fluorescent Dyes; D007476:Ionophores; D008563:Membrane Lipids; D009930:Organic Chemicals; D011744:Pyrimidinones; C402795:SYTOX Green; C003954:merocyanine dye