Detection and monitoring of normal and leukemic cell populations with hierarchical clustering of flow cytometry data

Flow cytometry is a valuable tool in research and diagnostics including minimal residual disease (MRD) monitoring of hematologic malignancies. However, its gradual advancement toward increasing numbers of fluorescent parameters leads to information rich datasets, which are challenging to analyze by standard gating and do not reflect the multidimensionality of the data. We have developed a novel method to analyze complex flow cytometry data, based on hierarchical clustering analysis (HCA) but with a new underlying algorithm, using Mahalanobis distance measure. HCA is scalable to analyze complex multiparameter datasets (here demonstrated on up to 12 color flow cytometry and on a 20‐parameter synthetic dataset). We have validated this method by comparison with standard gating approaches when performed independently by expert cytometrists. Acute lymphoblastic leukemia blast populations were analyzed in diagnostic and follow‐up datasets ( n = 123) from three centers. HCA results correlated very well (Passing–Bablok correlation coefficient = 0.992, slope = 1, intercept = −0.01) with standard gating data obtained by the I‐BFM FLOW‐MRD study group. To further improve the performance in follow‐up samples with low MRD levels and to automate MRD detection, we combined HCA with support vector machine (SVM) learning. HCA in combination with SVM provides a novel diagnostic tool that not only allows analysis of increasingly complex flow cytometry data but also is less observer‐dependent compared with classical gating and has potential for automation. © 2011 International Society for Advancement of Cytometry