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. Y_cav/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.