Increased hemolysis from saline pre-washing RBCs or centrifugal pumps in neonatal ECMO.

OBJECTIVES: Hemolysis is a significant complication of extracorporeal membrane oxygenation (ECMO), with a reported incidence of 12.2%. The aims of this study were (1) to investigate hemolysis caused by saline-washed versus unwashed RBCs, (2) to determine in vitro the effects of saline washing on erythrocyte hemolytic markers and (3) to investigate hemolysis by centrifugal versus roller pumps. STUDY DESIGN: (1) To evaluate the effect of pre-transfusion saline-washing versus non-washing, the peak plasma-free hemoglobin (FHb) and total bilirubin in the first 3 days versus the next 4 days of ECMO were compared (2) Pre- and postsaline-washed RBCs were analyzed for K+ hemoglobin, mean corpuscular volume, FHb and hemolysis at baseline and after 4 h of storage at 4 degrees C. (3) Over 10 000 neonatal ECMO cases were retrospectively reviewed to study the effect of pump type on hemolysis. RESULTS: (1) The washed blood group had significantly more hemolysis within the first 3 days of ECMO. (2) Immediately after saline washing, the K+ and Hb concentrations were significantly decreased compared with unwashed blood, and these differences were maintained after 4 h. The osmotic fragility of washed RBCs after 4 h of storage at 4 degrees C was significantly higher than at baseline. (3) Hemolysis was reported more often in the centrifugal than in the roller pump group. CONCLUSIONS: (1) Using unwashed RBCs decreased hemolysis within the first 3 days of ECMO. (2) Saline washing, while decreasing the concentration of K+ in the plasma, significantly increases RBC membrane osmotic fragility. (3) Hemolysis is linked to the use of centrifugal pumps.

Determination of heparin-induced thrombocytopenia: a rapid flow cytometric assay for direct demonstration of antibody-mediated platelet activation.

Heparin-induced thrombocytopenia (HIT) and thrombosis are serious complications of heparin therapy. Recently, we have reported a practical and rapid functional flow cytometric assay (FCA) for the diagnosis of HIT with high specificity and sensitivity compared with the radioactive serotonin-release assay (SRA). In the present study, we added an immune-neutralization assay to directly demonstrate the antibody-mediated process, and tested the immune compatibility of low-molecular-weight heparin (LMWH) Lovenox and the heparinoid Orgaran (danaproid) using plasma from 18 patients with HIT confirmed by both FCA and SRA. The clinical utility of this modified method is demonstrated by a pediatric patient with a complex clinical presentation who developed thrombocytopenia with multiple thromboses while on heparin therapy. ELISA and SRA (performed in three independent laboratories) for diagnosis of HIT were both negative. In contrast, the FCA for detecting activated platelets expressing anionic phospholipids, was highly and reproducibly positive with both unfractionated and LMWH. Another FCA also demonstrated the surface expression of the alpha-granule membrane p-selectin (CD62p). Compatibility testing with the heparinoid Orgaran was also positive (and with plasma from 4 of the 18 patients with HIT). Heparin was discontinued, along with full recovery of the platelet count. The capacity of the patient's plasma to activate platelets in the presence of heparin gradually decreased over 4 weeks consistent with antibody clearance. The responsible mechanism was clarified using an immune-neutralization assay, which showed a dose response neutralization of the plasma activity by antibodies against human Immunoglobulin G (IgG) and IgM. This assay was also reproducible in the 18 patients with HIT. We conclude that the functional FCA with its modification is practical, sensitive, and specific for reliable diagnosis of HIT. It can simultaneously assess the compatibility of alternative therapies and directly confirm the antibody-mediated process. Further, it is particularly useful to clarify mechanisms of thrombocytopenia and thrombosis and to direct therapy in patients with a complex presentation and confounding laboratory results who often need prompt diagnosis and treatment.