EX-VIVO ELUTION OF HEMODIALYZERS - AN ADDITIONAL CRITERION FOR THE ASSESSMENT OF BIOINCOMPATIBILITY

The analysis of hemodialysis (HD)-related bioincompatibility is focuse d mainly on phenomena observed in peripheral blood. However, since bio compatibility originates inside the dialyzer, white blood cells (WBC) adhering to the dialyzer are probably most subject to the influence of both dialyzer membrane and dialysate. In order to collect membrane-ad herent cells, a reliable and reproducible elution technique was develo ped. After 3 h of HD, blood was returned to the patient with 0.9% NaCl . Then, dialyzers were eluted by recirculation of phosphate-buffered s aline (PBS) or PBS/3 mM EDTA for 20 min, with or without prior flushin g with 200 ml PBS. Finally, remaining adherent cells were collected by an afterwash with 10% trypsin. These solutions, as well as blood samp les, were analyzed for WBC count, viability and differentiation. Rando m eluate samples were analyzed by flow cytometry, and the influence of elution on PMN activation was tested in a separate control experiment . WBC numbers decreased by flushing before elution, whereas cell numbe rs were maximal after elution with PBS/3 mM EDTA (30 x 10(6)). Trypsin afterwash resulted in a further yield of 12 x 10(6) cells. The eluate s contained 81% PMN (blood 68%, p < 0.01), with a degranulated appeara nce, and only 12% lymphocytes (blood 21%, p < 0.05); eel viability in the eluates was >95%. The eluted cells could be analyzed by flow cytom etry, and the procedure itself induced only minimal PMN activation. In conclusion, a maximal number of adherent cells, consisting mainly of PMN, was obtained by direct elution with PBS/3 mM EDTA. The method its elf did not induce marked PMN activation, and the cells obtained were suitable for further investigations, including flow cytometry.