title: Method validation for automated isolation of viable peripheral blood mononuclear cells.
abstract: 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 was considered optimal for further processing, allowing isolation of plasma and PBMCs from a single sample. The three gradients gave similar outcomes in terms of cell yield, viability, and WBC subpopulation distribution. Ficoll showed some advantages and was selected for further evaluations. Optimization of the automated protocol script using a CPT gradient gave 61% cell recovery. No significant differences in quality, quantity, and WBC subpopulation distribution were seen between the two freezing methods, and Mr. Frosty was selected. The manual and automated protocols were reproducible in terms of quantity, recovery, viability, WBC subpopulation distribution, gene expression, and LCL transformation. Most (75%-100%) of the 13 robustness parameters were accepted for both methods with an 8 h pre-centrifugation delay versus 38%-85% after 24 h. Differences identified between the automated and manual methods were not considered consequential.
  
  
  CONCLUSIONS
  We validated the first fully automated method for isolating viable PBMCs, including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection.
authors.affiliation: Integrated BioBank of Luxemburg (IBBL), Luxembourg.
authors.forename: Gael
authors.initials: G
authors.lastname: Hamot
authors.affiliation: Integrated BioBank of Luxemburg (IBBL), Luxembourg.
authors.forename: Wim
authors.initials: W
authors.lastname: Ammerlaan
authors.affiliation: Integrated BioBank of Luxemburg (IBBL), Luxembourg.
authors.forename: Conny
authors.initials: C
authors.lastname: Mathay
authors.affiliation: Integrated BioBank of Luxemburg (IBBL), Luxembourg.
authors.forename: Olga
authors.initials: O
authors.lastname: Kofanova
authors.affiliation: Integrated BioBank of Luxemburg (IBBL), Luxembourg.
authors.forename: Fay
authors.initials: F
authors.lastname: Betsou
mesh_terms: D001331:Automation; D001772:Blood Cell Count; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D005615:Freezing; D006801:Humans; D007962:Leukocytes / Q000166:cytology; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D015203:Reproducibility of Results