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

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 (P(aw)). Lung volume can change significantly after fixation as P(aw) change. To determine the variation of lung volume after fixation, we measured the volume of intact fixed lungs by serial immersion in saline (V(imm)) at selected time points, compared with measurements obtained by point counting [Cavalieri Principle (V(cav))] after tissue sectioning to release P(aw). V(imm) was systematically higher than V(cav) 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. V(imm) remained 14% higher than V(cav) in dog lungs even after pressure release. V(cav)/V(imm) was systematically lower in the upper than the lower strata of the same lung. We conclude that V(cav) measured on lung slices after relaxation of P(aw) more precisely represents the state of the tissue to be used for subsequent morphometric analysis, particularly for large lungs.