In vitro evaluation of neutrophil viability after exposure to a hypotonic medium

Separation techniques for radiolabelled leukocytes have inherent problems with contaminants (e.g. platelets and erythrocytes). Hypotonic lysis methods can eliminate the erythrocytes, but the question of neutrophil viability after an exposure to a hypotonic solution (i.e. sterile water) remains. Ficoll/ hypaque two-density gradient separation was performed on donor whole blood to obtain a pure neutrophil suspension. A timed sequence of water exposure was done for 5-100 s on the neutrophil preparations. The viability of these preparations was evaluated using flow cytometry and chemotaxis. The trypan blue staining method was used to document cell death. With water exposures ranging up to 100 s, 2.04 +/- 1.80% neutrophils exhibited cellular degradation by flow cytometry, and all samples demonstrated viable neutrophils by chemotaxis and trypan blue staining. The hypotonic medium exposure times for leukocyte separations should be less than 30 s for neutrophils to retain their viability by these in vitro techniques.