Method Validation for Automated Isolation of Viable Peripheral Blood Mononuclear Cells

Background: This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood, and compare the two methods. Methods: The manual method was optimized for whole blood centrifugation speed, gradient type (Ficoll, Leucosep, CPT), and freezing method (Mr Frosty, Controlled Rate Freezing). Various parameters of the automated protocol using a CPT gradient on a Tecan liquid handler were optimized. Optimal protocols were validated in parallel for reproducibility and robustness. Optimization and validation were assessed in terms of cell yield, viability, recovery, white blood cell (WBC) subpopulation distribution, gene expression, and lymphoblastoid cell line (LCL) transformation. Results: An initial centrifugation of whole blood at 2000 g...