Assessment of the hydration state of sickle cells by phthalate ester density distribution

Abstract

Intracellular hemoglobin S (Hb SS) concentration, a function of cell hydration, has a major influence on the rate of Hb SS polymerization and, therefore, cellular sickling. To determine the density distribution of homozygous sickle hemoglobin cells as a function of cell hydration, cells were incubated in autologous plasma buffer mixtures with final osmolalities ranging from 195 to 490 mosm/kg at ambient Po2. The density distribution of the cells was determined by differential flotation on 20 mixtures of di-n-butyl and dimethyl phthalates with specific gravities of 1.062 to 1.142. Mean cell hemoglobin concentration (MCHC) and mean cell volume (MCV) were determined by standard manual procedures. Cell shape was assessed by scanning electron microscopy (SEM), and the axial ratio (L/W) of the elliptical dense cell fraction measured by an image analyzer interfaced with a computer. The density distribution of normal red blood cells lies within a narrow 1.090 to 1.118 gm/ml density band with the middle or transitional 60% (T60) of the cells occupying a density range of 0.0067 +/- 0.0007 gm/ml (+/- SD). The density distribution of sickle cells shows a broader density band of 1.064 to 1.134 gm/ml, and the T60 was 0.0139 +/- 0.0022 gm/ml. The mean T60 did not change with osmotic variation but the mean T60 of Hb SS cells was significantly greater (P less than 0.005). MCHC and 1/MCV varied directly with the median density of the density distribution. By linear regression analysis and Ponder's osmotic equation, it is evident that sickle cells exhibit restricted volume increases in hypotonic media.(ABSTRACT TRUNCATED AT 250 WORDS)