Optimizing Manual Flow Cytometric Analysis for Lymphoma Using an Automated Computational Tool
Fiona E Craig, Ryan Brinkman, Stephen P Ten Eyck, Nima Aghaeepour. University of Pittsburgh School of Medicine, Pittsburgh, PA; British Columbia Cancer Agency, Vancouver, BC, Canada; University of British Columbia, Vancouver, BC, Canada
Background: Although many clinical laboratories are adopting higher color flow cytometric assays, the approach to optimizing data analysis remains subjective. To address the question “What is the best strategy to reliably distinguish reactive and neoplastic germinal center B-cells?” we applyed a computational tool that has demonstrated utility in cellular immunology. RchyOptimyx constructs cellular hierarchies by combining automated gating with dynamic programming and graph theory to provide the best gating strategies to identify a target population correlated with a desired outcome.
Design: Cases of reactive lymphoid hyperplasia and germinal center lymphoma, evaluated using the 8-color flow cytometric tube (CD45 V500, CD20 V450, kappa FITC, lambda PE, CD19 PE-Cy7, CD5 PerCP-Cy5.5, CD10 APC, CD38 APC-H7), were identified. List-mode data was analyzed using Rchy-Optimyx. This information was then used to refine manual analysis using DIVA software (BD Bioscience).
Results: Computational analysis identified cellular hierarchies with a high predictive power, including the phenotype CD19+CD10+CD38-, which was not emphasized in the previous analysis.
Manual reanalysis confirmed significantly higher CD10+/CD38- B-cells in lymphoma (median 12.44%, range 0.74 – 63.29, n=52) than reactive (median 0.24%, 0.03 – 4.49, n=48)(p=0.0001). Identifying a population of CD10+CD38- B-cells by visual inspection had a higher sensitivity for lymphoma than aberrant antigen expression, but was not entirely specific.
|Reactive (n=48)||Lymphoma (n=52)||Sensitivity||Specificity|
|Aberrant CD19, CD20 or CD10||0||25||52.1%||100%|
|Aberrant CD19, CD20 or CD10, or CD10+/CD38-||11||42||87.5%||75.0%|
|Ig light chain restriction||0||47||90.4||100|