LAUSR

THE importance of cytogenetic abnormalities in clinical diagnosis and response has been well established in particular for hematological malignancies with the diagnostic tools being microscopy-based karyotyping or fluorescence in situ hybridization (FISH). For chronic lymphocytic leukemia (CLL), the associated genetic abnormalities are heterogeneous with trisomy 12 (+12) among the most common (1) and 17p deletions (del(17p)) among the most adverse prognostic parameters for response and survival in CLL (2). Immunophenotypically, CLL cells are typically characterized by co-expression of CD5, CD19, CD20, and CD23 with low expression of surface immunoglobulin, CD20 and CD79b compared to normal B cells and malignant clone-specific kappa or lambda immunoglobulin light chain restriction (3). Following remission induction, flow cytometry plays an important role in the detection of minimal residual disease with sensitivity of detection in the range of 1 CLL cell in 10,000 leukocytes (0.01%) (3). In contrast, conventional slide-based FISH applied to detect chromosomal abnormalities typically has a sensitivity of 5–7%, several orders of magnitude lower than that of flow cytometric phenotyping. The development of suspension fluorescence hybridization approaches quantifiable by flow cytometry was first reported in the context of measuring telomere lengths (4). A drawback of this approach, however, particularly in the context of spot counting is that the number hybridization sites is being assessed only by fluorescence intensity on the premise that intensity is directly correlated with number of hybridization spots/sites. However, without specific fluorescence spatial information, this approach does not account for potential non-specific reactions outside the nuclear area of the cell. The development of imaging flow cytometers combined the high-throughput capability of flow cytometry with the capability of microscopy to provide high image content information on each individual event acquired. This technology thus had the potential to perform image analysis on rare cell populations in statistically robust cell numbers in principle limited only by the number of events acquired. The application of imaging flow cytometry to detect aneuploidy using a FISH in suspension protocol (5) demonstrated that spotcounting algorithms alone were insufficient to derive accurate results. The source of the inaccuracy was the occurrence of super-imposed hybridization spots related to the 2D projection of the 3D cells in suspension, which could lead to underscoring of the spot counts. Additional artifacts were overscoring of spot counts due to disaggregation of hybridization spots. It was determined and validated that these artificial spot count results could be corrected by incorporating a spot intensity measurement with a true single hybridization spot having 50% of the intensity of a spot that resulted from two superimposed spots and that the total intensity of a true trisomy had 1.5-fold the intensity of a true disomy (5). It was further demonstrated that sensitivity of detection of trisomy 8 in AML was as low as 0.5% (5). The first imaging flow cytometry application of FISH in suspension in CLL was