Homotypic interactions between CLL lymphocytes in culture and within tissues. Panel A. Phase-contrast image of live cells within culture showing that cytoplasmic projections extend directly between adjacent cells (arrowed). Panel B. Immunoperoxidase staining shows that within some small clusters CD4 cells are demonstrated, but these are absent in most cases. Panel C. Immunostaining of cell groups demonstrating the homotypic nature of interactions: left panel shows F-actin (Texas Red phalloidin) illustrating irregular cell shape and interactive projections connecting cells; centre panel cell nuclei (dapi); right panel lineage markers - in this example CD3-FITC to show that T lymphocytes are separate from the groups. Groups were demonstrated in all CLL cases tested (n = 8). The majority of groups contain 2–3 cells, but larger groups were also seen. Panel C. Lymph node tissue involved by CLL lymphocytes immunostained to demonstrate cell process formation (CD23-peroxidase/DAB – brown, counterstained with hematoxylin). Fine focal depth microscopy shows successive z-planes illustrating contact between adjacent CLL lymphocytes, and irregular cell shape with extended processes. Examples with extended cell processes passing through the tissue planes shown are specifically indicated (A-D arrowed on slide).