Because standard Ficoll gradient lymphocyte separation and cryopreservation are prohibitively expensive for large epidemiologic studies, investigators in the PLCO trial developed and validated a field-collection protocol29 involving the collection of blood at the nationwide screening centers in acid–citrate–dextrose, followed by express shipment to a processing laboratory in Frederick, Maryland, where whole-blood aliquoting with 5% dimethylsulfoxide to cryovials and controlled-rate step-freezing took place, to establish a trial-wide viable lymphocyte repository. Whole-blood samples (1 ml) that were stored in liquid nitrogen freezers were thawed and an aliquot of 500 μl was used for six-color flow cytometry (with antibodies CD45, CD19, CD5, CD10, kappa, and lambda); the remaining 500 μl of whole blood was used for analyses of the immunoglobulin heavy-chain variable (IGHV) gene.
