Flow cytometric analysis of CD34, Kit (CD117), and G-CSFR (CD114) expression of bone marrow cells.

One million cells were simultaneously incubated with FITC-labeled monoclonal anti-CD34, PE-conjugated anti–c-Kit, and biotin-conjugated anti–G-CSFR for 30 to 40 minutes at 4°C. Cells were then washed twice and stained with streptavidin labeled with allophycocyanin for 15 minutes at 4°C. After the addition of PI at a concentration of 1 μg/mL, cells were applied to FACS Vantage. The appropriate isotype controls, FITC-, PE-, and biotin-conjugated mouse IgG1a were used to identify background staining. More than 3 × 10⁵ events were collected and then analyzed. Low to medium forward scatter and low side scatter (A, R1) negative for PI fluorescence (B, R2), and positive for CD34 (C, R3) gates were used. The expressions of Kit and G-CSFR within gated cells are shown for representative subjects without SCN (D, E) and 5 patients with SCN (F, patient 1; G, patient 2; H, patient 3; I, patient 4; J, patient 5). Quadrant percentages are indicated in each cytogram. The quadrant percentages (mean ± SD) of Kit⁺/G-CSFR⁺, Kit⁺/G-CSFR⁻, Kit⁻/G-CSFR⁺, and Kit⁻/G-CSFR⁻ cells in CD34⁺ cells in 9 subjects were 33.4 ± 8.8, 16.1 ± 6.2, 26.4 ± 9.5, and 24.1 ± 13.6, respectively. Those from 5 patients were 5.9 ± 3.5, 15.4 ± 8.9, 45.2 ± 21.0, and 33.5 ± 13.6, respectively. The difference in the frequency of Kit⁺/G-CSFR⁺cells between subjects without SCN and patients with SCN was statistically significant (P < .001).