Following a succession of changes in circuitry and priming additives between 1993 and 1998, a comprehensive re-evaluation of neonatal cardiopulmonary bypass (CPB) practice was undertaken. Samples from 10 infants (Group 1) undergoing CPB were evaluated for osmolality, oncotic pressure, total protein, hematocrit, glucose, and electrolytes (Na+, K+, iCa2+). These samples were tested at six measurement points: (1) after priming, (2) patient pre-CPB, (3) CPB-start, (4) CPB-mid, (5) CPB-end, and (6) post-modified ultrafiltration (MUF). Prime volumes were also carefully measured as well as the type and amount of volume given during CPB. After evaluating the initial data, changes in protocol regarding mannitol, calcium correction, and oncotic strength on CPB were made. Following implementation of these protocol changes, a second set (Group 2) of 10 infants was identically evaluated. Group 1 prime osmolality was 379 +/- 44 mOsm/kg, while Group 2 prime osmolality was 324 +/- 14 mOsm/kg (p = 0.003). There were no differences in osmolality between groups during bypass and osmolality was unaffected by modified ultrafiltration. Ionized calcium levels were significantly different at the end of bypass between Group 1, 0.6 +/- 0.1 mmol/l; and Group 2, 1.17 +/- 0.24 mmol/l (p < 0.001). In Group 1, there was a 40% drop (p = 0.001) in colloid osmotic pressure (COP) levels from pre-CPB (13.3 +/- 3.4 mmHg) to CPB-end (8.8 +/- 1.2 mmHg). In Group 2, there were no differences in COP during CPB. COP levels of Group 1 and Group 2 at CPB-end were 8.8 +/- 1.2 mmHg and 14 +/- 1.9, respectively (p < 0.0001). Total volume addition during bypass for Group 1 was 363.5 +/- 148.7 ml and for Group 2 was 245.1 +/- 92.2 ml (p < 0.05). In conclusion, progressive changes in neonatal circuits and techniques can have potentially wide-ranging effects on electrolyte and osmotic/oncotic homeostasis. An audit of perfusion management through expanded laboratory tests is recommended, especially in periods of change.