Aggregation of monocytes and platelets interferes in measurement of monocyte viability with phosphatidylserine expression but not with mitochondrial membrane potential in whole blood

Abstract

Background: Bacterial lipopolysaccharides (LPS) induce cell death and procoagulant tissue factor (TF) in purified cultured human monocytes. Phosphatidylserine (PS)-expression on the outer membrane of monocytes following cell death contributes to increased procoagulant activity. LPS also induce TF on monocytes in whole blood (WB), but it is unknown whether LPS induce cell death. Activated platelets that aggregate with monocytes in non-EDTA anticoagulated WB also express PS and may potentially interfere in viability measurement on monocytes in WB. Methods: Viability of monocytes was estimated in purified monocytes and unlysed citrated WB incubated without and with LPS using two markers of early cell death; Lactadherin (PS-exposure) and Mitoprobe™ DilC (Mitochondrial Membrane Potential, MMP), and one late marker; Sytox Blue (membrane impermeant DNA stain). To study the interfering effect of monocyte-platelet aggregates (MPAs) on estimated monocyte viability, addition of EDTA or anti-CD62P was used to inhibit monocyte-platelet binding. Results: In WB, the median percentage of viable monocytes without and with EDTA was 21% and 93% with PS-exposure and 89% and 99% with MMP, respectively. Addition of EDTA reduced the percentage of MPAs (81% to 8.2%). Addition of anti-CD62P also reduced the percentage of MPAs (97% to 42%) and PS-expression on monocytes (MFI 28.9 to 3.9). LPS induced death of monocytes after 3 hours in purified monocytes, but not in WB. Conclusions: MPAs interfered in monocyte viability measurement in citrated WB with PS-exposure, but not with MMP. LPS induced death of monocytes in purified culture, but not in WB. © 2014 Clinical Cytometry Society.

Monocytes and macrophages in flow cytometry

“…On the other hand, apparent aberrant expression of normal monocytes can happen in case of cell aggregates and doublets must be excluded. However, platelets can adhere on monocyte's surface with negligible change on monocyte size that is not detected by usual way of doublet exclusion but brings unusual but non pathological labeling such as CD31 as shown by Amundsen et al (8). This paper also highlights the importance of pre-analytical conditions to ensure that cells are really analyzed individually, as single.…”