PAX5 Expression by Flow Cytometry. A Feasibility Study
Mihai Merzianu, Dalin Pan, Rosemary Furlage, Paul K Wallace. Roswell Park Cancer Institute, Buffalo, NY
Background: PAX5 is essential for B cells differentiation and maturation and PAX5 protein is widely used in clinical practice to establish B-lineage by immunohistochemistry (IHC). We assess here feasibility of PAX5 testing by flow cytometry (FCM) using a commercially available monoclonal antibody (mAb).
Design: Twenty cell suspensions from routine, unselected clinical samples (9 marrow, 8 lymph node, 2 tissue, 1 peripheral blood) were stained with PAX5 mAb (1H9, eBioscience, San Diego, CA) conjugated with APC using a FOXP3 protocol (eBioScience) after being surface stained with a panel including CD10 BV421, CD19 PECy5, CD34 PE and CD45 V500. Canto II (BD Biosciences) and WinList (Verity Software) were used for acquisition and analysis. A FMO control lacking the PAX5 antibody was run for each sample to establish positive and negative regions. PAX5 IHC (BD Biosciences) expression was semiquantitatively assessed in corresponding biopsies in quantiles.
Results: Six acute leukemias (3 B-, 1 T-, 1 myeloid, 1 mixed T-Mye), 8 B-non Hodgkin (B-NHL) and 1 classical Hodgkin (CHL) lymphomas and 5 benign samples were included. PAX5+ and CD19+ events averaged 47% (median 48, range 0.9-95) and 51% (median 52, range 0.7-95.5) of CD45+ cells, respectively. PAX5+CD19- and PAX5-CD19+ events averaged 3.3% (median 1.1; range 0.06-25.6) and 7.5% (median 2.9; range 0.05-77) of analyzed cells, respectively. PAX5 was expressed in all neoplastic (8 B-NHL and 3 B-ALL) and benign B cells (n=5), similar to CD19, but in none of the T and/or myeloid neoplastic cells (0/3). CHL tumor cells were not detected. Using a 10% threshold, sensitivity and specificity for detecting neoplastic B cells were 91% and 100%. Compared to IHC PAX5, PAX5+ events count by FCM was in the same or within 1 quintile in 8, higher in 2 and lower in 6 of 16 cases tested.
Conclusions: FCM PAX5 testing is feasible in clinical samples using a commercial mAb. This test could be informative when paraffin embedded tissue is not available or when solely FCM is utilized for phenotyping, and may be used in an add-on panel for selected cases, including acute leukemias with ambiguous, minimal or no differentiation.
Wednesday, March 6, 2013 1:00 PM
Poster Session VI # 214, Wednesday Afternoon