The measurement of lymphocyte volume: Importance of reference particle deformability and counting solution tonicity

Abstract

We have determined the influence of reference particle deformability and suspending buffer tonicity on the measurement of lymphocyte volume by an electronic particle volume analyzer. When the volume analyzer was standardized with latex spherules having a shape factor (f(e)) of 1.5, red cell volume was 96 cu μm and lymphocyte volume was 289 cu μm. The red cell volume corresponded closely to the true red cell volume; the true lymphocyte volume, however, was 218 cu μm when measured by the lymphocytocrit/lymphocyte count and 203 cu μm by wet lymphocyte weight and density (mean ~210 cu μm). The difference between the electronic volume (V(e)) of 289 cu μm and true lymphocyte volume of 210 cu μm was due to the influence of lymphocyte deformability (shape factor) as it traverses the sizing aperture. Since the true volume equals the V(e)/f(e), the red cells with a shape factor near 1.0 were sized appropriately by this method. In contrast, the lymphocyte shape factor was 1.38; thus, the true lymphocyte volume was 289 cu μm/1.38 or 210 cu μm. The tonicity of the suspending solution also influenced the measurement of particle volume when osmotically inactive standard particles (e.g., latex spherules) were used as a reference. Whereas the true lymphocyte volume was 210 cu μm at 286 mosmole/liter, it was 194 cu μm at 330 and 229 cu μm at 250 mosmole/liter. The standard counting solution, Isoton, is hyperosmolar (330 mosmole/liter) and causes an 8% shrinkage of osmotically active cells.