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Hemodialysis with new cellulosic membranes is associated with profound granulocytopenia, with a nadir 15 min after initiation, followed by a rebound leukocytosis seen 1 h after initiation and persisting up to the termination of dialysis. The rapid reversal of granulocytopenia during hemodialysis has previously been ascribed to down-regulation of granulocyte C5a receptors. In this report, a method of characterizing C5a receptors by using a novel probe consisting of C5a attached to biotin via a six-carbon spacer chain is described. Cellulose acetate electrophoresis and cation exchange HPLC demonstrated a biotin-to-C5a ratio of 1:1. Analysis of granulocyte cell surface C5a receptors were performed with the probe with a fluorescein-avidin conjugate and by using fluorescence flow cytometry. The maximum decrease in C5a receptors was measured at the 15-min sampling time, when the number of C5a receptor decreased from 189,240 +/- 24,500 predialysis to 160,740 +/- 19,380 receptors (P was not significant) at the nadir of granulocytopenia. However, during recovery from neutropenia, granulocyte cell surface C5a receptors increased to 172,140 +/- 19,380 at 30 min and 193,800 +/- 24,510 at the end of dialysis. Concentrations of C3a and C5a peaked at 15 min and declined rapidly thereafter, but both remained significantly above baseline at all times. These studies suggest that down-regulation of C5a receptors, which is seen maximally at 15 min after initiation of dialysis, does not sufficiently account for the reversal of granulocytopenia during hemodialysis.