Figure 6. Inhibition of CDC by low pH occurs in whole blood and is a reversible phenomenon.

A and B. Alemtuzumab-treated PBMCs (5 × 10⁵/100 μl) were incubated for 30 min at 37°C with 10% autologous HS, at pH 5.5. Then, the pH was adjusted to pH 7.3 (grey histograms) or remained at pH 5.5 (black line), and cells were further incubated for a period of 30 min at 37°C. Finally, necrosis was evaluated by PI staining and flow cytometry. Controls represent alemtuzumab-treated PBMCs incubated for 60 min at 37°C and pH 7.3 in the presence of 10% autologous HS (dotted lines). Representative histograms or the mean ± SEM of 5 experiments are showed (*p<0.05 vs pH 7.3). C and D. Aliquots of peripheral blood (100 μl) collected on 3.2% sodium citrate (C) or bivalirudin (50μg/ml) (D) were adjusted to different pH values and treated with alemtuzumab (50 μg/ml). After 30 min incubation at 37°C, cells were stained with FITC-conjugated anti-CD3 mAb and PerCP-7AAD. The percentage of CD3⁺/7AAD⁻ cells was determined for each condition and the results were expressed as the percentage of T cell depletion compared with untreated blood samples. Controls were made by incubating aliquots of peripheral blood, adjusted to different pH values, without alemtuzumab. Data represent the mean ± SEM of 4-5 experiments. *p<0.05 vs pH 7.3.