Characteristics of leucocyte adhesion directly observed in flowing whole blood in vitro

Use of whole blood in adhesion assays allows analysis of the rheological an d haematological factors that may influence adhesion, and avoids the need f or isolation procedures that may modify the properties of leucocytes. We ha ve adapted an in vitro flow model to allow videomicroscopy of leucocytes fl uorescently labelled with rhodamine 6G (20 mug/ml) in anticoagulated whole blood. Blood was perfused at a range of wall shear rates (35-280/s) through a vertical glass capillary with a rectangular cross-section (microslide) t hat had been coated with P-selectin (10 mug/ml). Nearly all adherent cells were rolling in blood that had been anticoagulated with buffered citrate, b ut 40-50% became immobilized when heparin or thrombin inhibitor (PPACK) wer e used. The efficiency of leucocyte adhesion decreased steadily during 1-4 h of blood storage. Smaller fluorescent cells (lymphocytes) adhered less ef ficiently than larger cells (granulocytes) and rolled faster. Adhesion decr eased monotonically with increasing wall shear rate or stress, but the velo city of rolling varied little. Among healthy volunteer donors, adhesion cor related with blood leucocyte count, but did not vary significantly with nat ural variation in haematocrit, blood viscosity or red cell aggregation. In conclusion, we have characterized adhesion of leucocytes in flowing whole b lood, identified key experimental variables and demonstrated that physical environmental factors can markedly influence adhesive behaviour.