Immune reconstitution and alemtuzumab pharmacokinetics. (A) Early and long-term lymphoid recovery. Median absolute total lymphocyte counts with 12 months follow-up for the high-dose in vivo (RIT) group and 6 months follow-up for the low-dose in vitro (myeloablative allograft) group. (B) Recovery of T-cell subsets. Median lymphocyte counts after transplantation until 9 months after transplantation follow-up for the high-dose in vivo (RIT) group and 6 months after transplantation for the low-dose in vitro (myeloablative allograft) group. (C) Median serum alemtuzumab levels. Assays of alemtuzumab in patient serum: serum samples for pharmacokinetic analysis were collected 24 hours prior to the first infusion of alemtuzumab, 15 minutes before and after each infusion, 4 hourly for 24 hours after the last infusion, daily for the next 7 days, and then twice weekly until at least 28 days after the last alemtuzumab infusion. Sera were stored at -70°C until analysis. Before analysis, complement was inactivated by heat incubation of the samples at 56°C for 30 minutes. Validation studies have previously confirmed that this step does not alter alemtuzumab activity. Alemtuzumab activity was measured by indirect immunofluorescence as described in detail elsewhere.7,8 Previous studies have determined the following parameters for this assay18 : limit of detection, 0.3 μg/mL; analytical range, 0.5 to 20 μg/mL; linearity, 0.999; overall precision, 13%; and bias, +9%. There was no interference by a range of normal and patient control sera, and no reactivity with F(ab′)2 fragments of alemtuzumab. The assay has been shown to be robust with respect to variations in sample treatment and storage conditions, and variations in assay procedures. All sera samples were tested in duplicate at a final dilution of 1:2. Patient weight, total leukocyte count, underlying disease, and disease status at time of transplantation did not affect alemtuzumab levels or lymphocyte depletion.
