Density Gradient Centrifugation: A Method to Isolate CLL Cells from Peripheral Blood

- In chronic lymphocytic leukemia, or CLL patients, B cell lymphocytes significantly increase in the bone marrow and blood. To isolate these CLL cells from a blood sample, first, treat the sample with a human B cell enrichment cocktail that separates B cells using negative selection. The antibodies in the cocktail specifically recognize and cross-link the antigens expressed by non B cells and erythrocytes, forming dense complexes.

Next, layer the sample over a density gradient medium having a density optimal for lymphocyte isolation. With subsequent centrifugation, the different blood components separate based on variations in their densities relative to the gradient medium. The RBC-non-B-cell complexes with higher densities settle down through the medium, forming a pellet. The least dense plasma fraction forms the topmost layer. The less dense CLL cells form a white layer at the plasma-density medium interface.

Carefully transfer the white layer into a fresh tube. Centrifuge again to obtain a pellet containing purified CLL cells. In the following protocol, we show the isolation of chronic lymphocytic leukemia cells, from patient blood samples.

- Obtain peripheral blood samples fro