Human WAS B cells fail to form large homotypic aggregates and long protrusions. Peripheral blood B cells from WAS patients, healthy controls, or the WASI294T patient were stimulated with IL-4 and anti-CD40 mAbs for 5 days. (A) The extent of B-cell aggregation was assessed on fixed cells and representative images captured using an inverted microscope. Arrows indicate aggregates. (B) Quantitative determination of mean aggregate diameter. Per group, 5 to 20 aggregates were measured. (C) Electron microscopy of activated B cells. Representative images are shown. (D) Quantitative determination of membrane protrusions. Images of 30 individual cells in each group were captured under the electron microscope. The total membrane length of long (longer than 1.6 μm) and short (shorter than 1.6 μm) protrusions was measured. The measurements were done to cover the entire length of each protrusion, as explained in the image above panel D. Each symbol represents the relative amount of membrane covering long or short protrusions in a particular cell. Brackets indicate a significant difference of P < .001 using a 2-tailed t test.