Simultaneous Analysis of Phenotype and Cytogenetics Using Imaging Flow Cytometry: Time to Teach Old Dogs New Tricks

Abstract

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 demonstr...

Imaging flow cytometric detection of del(17p) in bone marrow and circulating plasma cells in multiple myeloma

“…This novel single cell cytogenetics approach therefore adds to the growing literature on ‘liquid biopsies’ for myeloma, such as quantification using ‘next generation’ flow cytometric methods 6,7,21 . It adds a new dimension enabling del(17p) to be detected in circulating plasma cells, even when only present in exceedingly small numbers 13,22 . By incorporating genetic analysis of the plasma cells, it surpasses the information obtained by standard flow cytometric cell enumeration alone.…”

“…6,7,21 It adds a new dimension enabling del(17p) to be detected in circulating plasma cells, even when only present in exceedingly small numbers. 13,22 By incorporating genetic analysis of the plasma cells, it surpasses the information obtained by standard flow cytometric cell enumeration alone. Currently 2% circulating plasma cells is recognized as a powerful predictor of prognosis in myeloma.…”

Imaging flow cytometry-based multiplex FISH for three IGH translocations in multiple myeloma

“…In addition, the use of unattached spherical moving cells in solution may also increase the incidental overlap of different fluorescent signals in the imaging flow cytometry system. Similarly, the problem of “2D projection of 3D cell” also causes the false-negative signal, as this also causes the incidental overlap of the same fluorescent signals, and, again, this error could increase by examining more hybridization signals in spherical moving single cells [ 41 ]. In addition, a higher signal-to-noise ratio is required for the accurate detection of a positive signal in the imaging flow cytometry, while the detection of the real signal could be sometimes low with moving cells in solution.…”