Typical flow cytometry analysis of IL-1β (A) and TNF-α (B) in peripheral blood monocytes from 3 pairs of patients with sickle disease and normal subjects.

Leukocytes were obtained from blood after the lysis of RBCs. Leukocytes were fixed and permeabilized; then they were immunostained for the expression of CD14, IL-1β, and TNF-α. Immunostained monocytes were analyzed by flow cytometry. A gate was placed around the mononuclear leukocytes based on their forward and side scatter. Monocytes within that gate were identified based on their expression of CD14-specific immunofluorescence. The x-axis measures mean fluorescence intensity of monocyte TNF-α or IL-1β, and the y-axis measures the cell counts. Each of the 3 graphs shows data from a pair of normal (white under the curve) and sickle (black under the curve) subjects that were collected and processed at the same time. (C) Sickle monocytes are activated: summary of IL-1β and TNF-α flow cytometry results. Expression of monocyte CD14, IL-1β, and TNF-α was measured by flow cytometry in 6 pairs of patients with sickle cell disease and normal subjects, as described in Figure 8A,B. Results are presented as the mean fluorescence intensity of monocyte IL-1β and TNF-α in patients and normal subjects. Mean fluorescence intensity of monocyte IL-1β was 13.3 in patients and 9.9 in normal subjects (P = .002). Mean fluorescence intensity of monocyte TNF-α was 13.4 in patients and 5.6 in normal subjects (P = .002). Data were normalized before statistical analysis. Five of 6 patients were in the hospital for sickle pain crisis.