The effect of anticoagulants on white blood cell l-selectin levels /

Abstract

Cell Adhesion Molecules (CAM's) are proteins embedded in the membranes of cells that bind to carbohydrates found on other cells. During an immune response, CAM's mediate the initial attachment of cells to the vessel wall. Tethering and "slow rolling" of the cell through the blood vessel occurs next, followed by movement of the cell through the vessel wall to the site of infection. L-selectin is a specific CAM that is initially involved in attachment of the white blood cell to the vessel wall. In order for the cell to squeeze through the blood vessel and migrate towards the site of infection, L-selectin must be shed. Stimulation of the white blood cells can be monitored by quantitating L-selectin levels on the cells. To study the white blood cells, it is important to use an anticoagulant that will prevent clotting of the blood, but not stimulate the cells. These studies compare the effects of four well known anticoagulants; EDTA, Potassium Oxalate, Sodium Citrate and Heparin on white blood cell expression ofL-selectin molecules. Blood was drawn from 7 volunteers into a vacuum tube containing one of the anticoagulants. Blood samples were removed at various timepoints up to one hour after collection and placed on ice. At one hour the cells were incubated with fluorescently labeled antibodies that bind specifically to L-selectin. Lysis buffer was added to lyse the red blood cells, leaving only the white blood cells. Paraformaldehyde was used to preserve the cells until they could be analyzed using a flow cytometer. The flow cytometer counts the cells one at a time using light patterns that deflect off of the cells. The fluorescence associated with the cells was measured to determine the L-selectin levels. The study showed that incubation in EDTA caused the least stimulation ofthe cells over time and therefore was the best anticoagulant to use when studying L-selectin.