Variation of lung volume after fixation when measured by immersion or Cavalieri method

Abstract

Organ volume is a critical parameter in morphometric analysis. The special problems of the lung as a nonsolid organ are overcome by tracheal instillation of fixatives at a constant airway pressure (Paw). Lung volume can change significantly after fixation as Paw change. To determine the variation of lung volume after fixation, we measured the volume of intact fixed lungs by serial immersion in saline (Vimm) at selected time points, compared with measurements obtained by point counting [Cavalieri Principle (Vcav)] after tissue sectioning to release Paw. Vimm was systematically higher than Vcav by 25% in dog lungs and 13% in guinea pig lungs ( P = 0.0003 between species). This size-dependent variability reflects residual elastic recoil, refolding and/or crumpling of alveolar septa after fixation. Vimm remained 14% higher than Vcav in dog lungs even after pressure release. Vcav/Vimmwas systematically lower in the upper than the lower strata of the same lung. We conclude that Vcav measured on lung slices after relaxation of Paw more precisely represents the state of the tissue to be used for subsequent morphometric analysis, particularly for large lungs.