Measurements of blood cell counts, coagulation factors, and blood gases
Arterial blood samples were obtained every 5 min to count blood cells using an Erma PCE 170 haematology analyser (Erma, Tokyo, Japan). Coagulation factors and blood gases were measured every 15 min. Hb concentration in the blood containing HbVs could not be accurately determined using the current analyser because liposome capsules interfered with the spectrophotometric measurement of Hb absorbance. The actual Hb concentrations were estimated using a previous method17. To measure plasma antithrombin (AT) III activity, prothrombin time (PT), and activated partial thromboplastin time (APTT), blood samples were collected into a heparinised syringe and centrifuged at 50,000 ×g at 4°C for 20 min to remove HbV particles and were measured at the Sanritsu Zelcova Laboratory (Tokyo, Japan). Plasma fibrinogen levels were measured using rabbit fibrinogen enzyme-linked immunosorbent assay kit (LifeSpan BioSciences, Seattle, USA). Blood gas analyses, including plasma lactate and arterial oxygen content (CtO2) levels, were performed using an ABL 80 blood gas analyser (Radiometer, Copenhagen, Denmark).