Background: Hemolysis, defined as the rupture of red blood cells (RBCs) with the release of hemoglobin into plasma, is a common concern during blood collection and extracorporeal circulation. Although excessive negative pressure has often been implicated as a contributing factor, the extent to which static negative pressure alone induces hemolysis remains unclear. This study aimed to quantitatively evaluate hemolysis under excessive static and repeated negative pressure conditions to clarify their respective roles in red blood cell damage.

Methodology: Pooled bovine blood was used to ensure uniform baseline conditions across all protocols. Two experimental conditions were examined: (i) static negative pressure at approximately −713 to −720 mmHg for 30 and 60 minutes, and (ii) repeated negative pressure loading with ten cycles alternating between near-vacuum (approximately −690 mmHg) and atmospheric pressure. Free hemoglobin (fHb) concentrations were measured spectrophotometrically using a commercial assay kit. Results below the limit of detection (LoD = 0.03 g/dL) were considered non-quantifiable and marked as “<0.03”.

Results: Under static negative pressure, mean fHb levels remained low (30 min: 0.040 ± 0.0071 g/dL; 60 min: 0.044 ± 0.0055 g/dL; n = 5 each). Repeated loading produced comparable values (0.050 ± 0.000 g/dL; n = 5), with no evidence of cumulative hemolysis. A transient detection near the LoD at 30 minutes was not sustained at 60 minutes, suggesting minimal, short-lived mechanical stress. Overall, neither static nor repeated negative pressure exceeding −700 mmHg caused meaningful hemolysis progression.

Conclusions: The findings support the theoretical understanding that blood behaves as an incompressible fluid, equilibrating hydrostatic pressure across the red blood cell membrane to prevent rupture under static vacuum conditions. These results indicate that dynamic factors, such as shear stress and cavitation during rapid aspiration or pump operation, rather than static negative pressure, are the primary contributors to hemolysis. This study provides evidence-based guidance for safer blood collection practices and the design of medical devices, emphasizing control of dynamic stresses rather than avoidance of static negative pressure.