Real time observations of polylysine, dextran and polyethylene glycol induced mutual adhesion of erythrocytes held in suspension in an ultrasonic standing wave field

Abstract

A technique which enables cells to be observed in suspension for times of the order of minutes (employing acoustic radiation foreces in a 1 MHz ultrasonic standing wave field) is described. Video recordings of the mutual adhesion of human erythrocytes in suspension have been analysed. Concave-ended cell doublets and linear rouleaux developed in 0.5–1.5% w/v Dextran T500 by a gradual (2.5–17 s) increase in the area of cell contact over the cell cross-section. The concave-ended rouleaux form was not seen in polylysine or in polyethylene glycol. In 5–7% dextran and in 20μg/ml polylysine mutual adhesion was a two stage process. Cells first form a strong local contact which persists (without apparently growing in area) for a number of seconds following which the cell surfaces move suddenly to form a spherical doublet. The average initial contact time and engulfment time for cells in 7% Dextran T500 are 18 and 2.7 s, respectively. The corresponding values for cells in 20 μg/ml, 14 kDa, polylysine are 2.7 and 0.3 s. There was no initial contact delay during spherical doublet formation in mg/ml polylysine. Electron microscopy showed that the intercellular seam for spherical doublets formed with all three agglutinating molecules was bent in a wavy (λ∼4 μm) profile. The thickness of the intercellular space varied in a spatially periodic way (λ∼0.8 μm) for cells in polylysine. Examples of periodic intercellular spaces were seen by light microscopy in polyethylene glycol induced clumps. The role of interfacial instability in the adhesion processes is discussed.