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

Background Determination of canine lymphomas immunophenotype by flow cytometry (FC) has become a routine analysis. Besides lymph nodes (LN), also peripheral blood (PB) and bone marrow (BM) samples are analyzed in order to stage the disease. Recently, a prognostic relevance for BM infiltration assessed by FC has been demonstrated in dogs with large B-cell lymphoma (LBCL). However, no data about analytical performances of FC in detecting neoplastic large B-cells in PB and BM are available. Aim of the present study is to define accuracy, sensitivity and precision of this technique, and to establish a cutoff suitable to safely discriminate between infiltrated and non-infiltrated PB and BM samples. Materials and methods Decreasing amounts of neoplastic cells from canine LNs with LBCL 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 CD21+ cells out of total CD45+ events were determined using a BD Accuri C6 flow cytometer. Agreement between expected and obtained percentages was evaluated via Passing-Bablok regression analysis. The CVs of ten acquisitions of the 0%, 1%, 3% and 10% dilutions were calculated to assess the intra-assay precision. ROC curves were drawn to identify the cutoffs most suitable to discriminate between PARR-positive and negative PB and BM samples. Results Optimal analytical accuracy and precision were achieved, with only a slight proportional error in BM dilutions. 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 0.77% best discriminated between infiltrated and non-infiltrated PB and BM samples, respectively, with >90% sensitivity and specificity. Conclusions Quantification of large B-cells in PB and BM samples by FC is reliable and has great analytical performances. Assessment of analytical performances of different instruments is warranted.