Venous blood into EDTA or ACD drawn immediately pre- and 24±4 hours post- infusion was evaluated by flow cytometry for adhesion markers on CD16b+ (ID3, Beckman Coulter) neutrophils: activated Mac-1 (CBRM1/5, eBioscience) or activated β2 integrin (ab13219, abcam), Mac-1 (ICRF44, BD Pharmingen), CD44 (G44-26, BD Pharmingen), E-selectin-Fc chimera (R&D Systems), L-selectin (DREG-56, eBioscience), LFA-1 (HI111 BD Pharmingen), and PSGL-1 (KPL-1, BD Pharmingen). Activated Mac-1 Ab detects the functionally active form of Mac-1(was performed on 200–800 mg/kg cohorts) and activated β2 integrin recognizes the high affinity conformation of β2 integrin, the β subunit of LFA-1 and Mac-1 (was performed on 100 mg/kg cohort).
With late afternoon or evening study drug administration, samples were stored overnight as needed (range 8–24 hours) at 4°C prior to analysis. When stored overnight, all paired pre-IVIG and 24 hour post-IVIG samples per patient were stored for uniform lengths of time. No adhesion markers except activated Mac-1 show expression increase at 24 hr compared to immediate analysis (Figure 1B ). Since the time interval to analysis was constant for each subject’s two samples, the ratio between the two samples (Figure 1A ) was unaffected by storage.
With late afternoon or evening study drug administration, samples were stored overnight as needed (range 8–24 hours) at 4°C prior to analysis. When stored overnight, all paired pre-IVIG and 24 hour post-IVIG samples per patient were stored for uniform lengths of time. No adhesion markers except activated Mac-1 show expression increase at 24 hr compared to immediate analysis (