Dialysis neutropenia is the result of pulmonary sequestration of neutrophils after complement activation by the dialyzer membrane. Increased expression of neutrophil adhesion receptors, such as CD11b/CD18, suggests that neutrophil adhesion to the capillary endothelium is a possible mechanism. An alternative hypothesis is that the complement fragment C5a modulates neutrophil mechanical properties via the cytoskeleton-largely filamentous actin (F-actin)-stiffening them and thereby slowing their passage through the pulmonary capillaries. To investigate this hypothesis, we developed an assay to measure the F-actin content of neutrophils in whole blood using flow cytometry and the stain NBD-phallacidin. We measured neutrophil F-actin content during hemodialysis of patients with polysulfone (N = 6), Hemophan (N = 6), and Cuprophan membranes sterilized with either ethylene oxide (N = 5) or steam (N = 6). Cell counts, neutrophil and monocyte CD11b expression and plasma C5a concentrations were also measured. The results confirm the strong relationship between the degree of neutropenia, increases in CD11b expression and plasma C5a levels reported by previous researchers. Modulation of the F-actin content of neutrophils was also strongly related to C5a levels, indicating that the neutrophil cytoskeleton is active during dialysis. Modeling of cell counts suggests that with Cuprophan a substantial fraction of neutrophils and monocytes are sequestered before they even pass through the dialyzer, suggesting some form of systemic activation of these cells. Evidence for systemic activation was also seen in measurements of F-actin content, but not CD11b expression, a finding that strengthens the case for the involvement of the cytoskeleton in dialysis neutropenia.
The Gradiflow (Life Therapeutics, Frenchs Forest, Australia) system is a novel electrophoretic technique that uses the dual characteristics of size and charge to separate target macromolecules from complex biological solutions. The system has the potential to selectively remove a range of moieties from blood and plasma in an in vivo system such as hemodialysis or, alternatively, in an in vitro setting such as a blood bank. In this study, the safety of a scaled down Gradiflow prototype device with a membrane surface area of 16 cm2 was investigated using an ex vivo ovine model. Physiologic, hematologic, and biochemical parameters were assessed in 12 sheep: 6 animals treated using the Gradiflow and 6 controls. The effects of the Gradiflow procedure on both whole blood and plasma were analyzed. The Gradiflow procedure was well tolerated, and the application of an electrical potential or exposure to the Gradiflow membrane did not cause any significant changes in the parameters measured. Hemoglobin levels remained stable in all groups during the 4-hour experiment. An early neutropenia was observed in all groups, although this appeared to be more pronounced with exposure to a plasma filter; the presence of the Gradiflow component had no separate influence. One sheep in the plasma group experienced septic shock, caused by Staphylococcus contamination of the separation membrane. Overall, the results indicate that there were no gross physiologic, hematologic, or biochemical adverse reactions to the ex vivo Gradiflow procedure.
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