Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation

Abstract

When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-microns glass pipette with a high negative aspiration pressure (delta P = -3.9 kPa), held in the pipette for 30 s (holding time, th), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in th. The echinocytes recovered spontaneously to discocytes in approximately 10 min, and there was no significant difference in recovery time at 20.9 degrees C, 29.5 degrees C, and 37.4 degrees C, respectively. At 11 degrees C the recovery time was significantly elevated to 40.1 +/- 6.7 min. At 20.9 degrees C the shape recovery time varied directly with the isotropic RBC tension induced by the pipetting. Sodium orthovanadate (vanadate, 200 microM), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 microM) reversed the pipette-induced echinocytic shape to discocytic in < 2 min, and the RBC became a spherostomatocyte-II after another 30 min. It was hypothesized that the increase in cytosolic pressure during the pipette aspiration induced an isotropic tension in the RBC membrane followed by a net inside-to-outside membrane lipid translocation. After a sudden release of the aspiration pressure the cytosolic pressure and the membrane tension normalized immediately, but the translocated phospholipids remained temporarily "trapped" in the outer layer, causing an area excess and hence the echinocytic shape. The phospholipid translocase activity, when not inhibited by vanadate, caused a gradual return of the translocated phospholipids to the inner layer, and the RBC shape recovered with time.

Mechanical and failure properties of single attached cells under compression

“…in excess of 50%. These bulges were also reported in studies involving micropipette aspiration and amphiphile-induced shape alterations in erythrocytes (Artmann et al, 1997;Ha¨gerstrand et al, 2000). In these studies, erythrocytes changed from a normal, discocytic shape to a spiculated, echinocytic shape.…”

“…This phenomenon might be attributed to a local increase of the area difference between the inner and outer lipid layer . In a similar manner to the present study, the erythrocytes in the micropipette aspiration experiments were compressed, leading to an increased lateral tension in the membrane (Artmann et al, 1997). This would induce a membrane phospholipid translocation from the inner to the outer lipid layer, thereby increasing the local area difference of the inner and outer lipid layer.…”

Effects of Salicylic Acid Derivatives on Red Blood Cell Membranes

“…echinocytic red blood cell shapes. The absolute amount of molecules deposited in the outer layer of the membrane may be sparse, since a small number of molecules incorporated asymmetrically can cause considerable shape changes (Ferrell et al 1985;Fuji et al 1979;Schrier et al 1992;Artmann et al 1997).…”

“…After reaching an assumed "saturation" with salicylamide in the outer leaflet at 5mM, salicylamide molecules may translocate to the inner leaflet, thus, partially compensate the total bilayer bending leading to a lower degree of echinocytosis at higher concentrations. Additionally, salicylamide may trigger a lipid flip-flop to minimize the lateral pressure differences induced by an asymmetric insertion of salicylamide molecules into the two leaflets as observed with the stomatocytogenic substance chlorpromazine (Schrier et al 1992;Artmann et al 1997).…”

Structure of a micropipette-aspirated vesicle determined from the bending-energy model

“…For years, the method has been used to experimentally probe and characterize the mechanical properties of cells, lipid-membrane vesicles, and even double emulsion-templated polymersomes (see, e.g., Refs. [2][3][4][5][6][7][8][9][10][11][12][13]). In addition, pipette aspiration is a popular method used to hold a vesicle in place in applications of other recent experimental techniques such as optical trapping [14][15][16].…”