title: Analytical and diagnostic validation of a flow cytometric strategy to quantify blood and marrow infiltration in dogs with large B-cell lymphoma.
abstract: Lymph node (LN), peripheral blood (PB), and bone marrow (BM) samples are commonly analyzed by flow cytometry (FC) for the immunophenotyping and staging of canine lymphomas. A prognostic value for FC BM infiltration in dogs with large B-cell lymphoma (LBCL) was demonstrated. Aim of this study was to define the analytical performances of this technique, and to establish a cutoff suitable to safely discriminate between infiltrated and noninfiltrated PB and BM samples.
  
  
  
  Large B-cells were added to control PB and BM samples, to achieve twelve different large B-cells concentrations, ranging from 0 to 50%. The percentage of large B-cells was recorded for each dilution, using a BD Accuri C6 FC. Accuracy was evaluated by Passing-Bablok regression analysis. Intra-assay precision was assessed at 0%, 1, 3, and 10% dilutions evaluating the CVs of 10 repeated acquisitions. ROC curves were drawn to identify the cutoffs most suitable to discriminate between 25 infiltrated (PARR-positive) and 25 noninfiltrated (PARR-negative) PB and BM samples, respectively.
  
  
  
  Optimal analytical accuracy and precision were achieved. Almost all CVs were <10%. Negative controls had up to 0.5% large B-cells, with 50 and 22% CV in PB and BM samples, respectively, 0.56 and 2.45% cutoffs were selected based on the ROC curves for PB and BM samples, respectively.
  
  
  
  Quantification of large B-cells in PB and BM samples by FC is reliable and analytical performances met the acceptance criteria. Assessment of performances of different instruments and protocols is warranted. © 2016 International Clinical Cytometry Society.
authors.affiliation: Department of Veterinary Sciences, University of Turin, Grugliasco, Turin, Italy.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Riondato
authors.affiliation: Department of Veterinary Sciences, University of Turin, Grugliasco, Turin, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Miniscalco
authors.affiliation: Department of Veterinary Sciences, University of Turin, Grugliasco, Turin, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Poggi
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Padua, Italy.
authors.forename: Arianna
authors.initials: A
authors.lastname: Aricò
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Padua, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Aresu
authors.affiliation: Department of Veterinary Sciences and Public Health, University of Milan, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Comazzi
authors.affiliation: Department of Veterinary Sciences and Public Health, University of Milan, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Martini
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000473:pathology; D001853:Bone Marrow / Q000473:pathology*; D004285:Dogs; D005434:Flow Cytometry / Q000379:methods*; D016130:Immunophenotyping / Q000379:methods; D008198:Lymph Nodes / Q000473:pathology; D016403:Lymphoma, Large B-Cell, Diffuse / Q000175:diagnosis* / Q000473:pathology*; D011379:Prognosis
keywords: dog; flow cytometry; large B-cell lymphoma; staging; validation