Analytical and diagnostic validation of a flow cytometric strategy to quantify blood and marrow infiltration in dogs with large b-cell lymphoma

Background 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 flow cytometric BM infiltration in dogs with large B-cell lymphoma (LBCL) was demonstrated. Aim of the present study was to define the analytical performances of this technique, and to establish a cutoff suitable to safely discriminate between infiltrated and non-infiltrated PB and BM samples. Methods 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 flow cytometer. Accuracy was evaluated by Passing-Bablok regression analysis. Intra-assay precision was assessed at 0%, 1%, 3% and 10% dilutions evaluating the CVs of ten repeated acquisitions. ROC curves were drawn to identify the cutoffs most suitable to discriminate between 25 infiltrated (PARR-positive) and 25 non-infiltrated (PARR-negative) PB and BM samples, respectively. Results 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. Conclusions 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. This article is protected by copyright. All rights reserved.