Image cytometry‐based detection of aneuploidy by fluorescence in situ hybridization in suspension

Abstract

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy‐based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry‐based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH‐IS), which enables the automated analysis of several log‐magnitude higher number of cells compared with the microscopy‐based approaches. This study demonstrates the applicability of FISH‐IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R2 = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH‐IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects. © 2012 International Society for Advancement of Cytometry

Fluorescent In Situ Hybridization in Suspension by Imaging Flow Cytometry

“…This protocol can routinely analyze orders of magnitude higher numbers of cells than conventional FISH, and the analysis can quickly identify chromosomal abnormalities. The lower threshold of sensitivity of FISH-IS is at least 1%, as previous described (11). This makes it ideal for the detection of abnormalities in rare cells, and therefore may improve the detection of MRD at clinical remission.…”

“…Design a spot mask for the SpectrumGreen signal (Figure 4). Optimize the radius and spot-to-background ratio of the mask on one cell but always check the efficiency of the mask on a selection of other cells to ensure the mask performs well for them all (11). …”

“…Its feasibility was previously described using Trisomy 8 in acute myeloblastic leukemia as a model (11). This protocol can routinely analyze orders of magnitude higher numbers of cells than conventional FISH, and the analysis can quickly identify chromosomal abnormalities.…”

“…Spiking a disomy sample with a monosomy should result in a measurable different distribution with a 2 fold difference in mean fluorescence E.g., taking male and female healthy donor samples and preparing each sample with CEP X will result in controlled monosomies and disomies of X (11). …”

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

“…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). 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.…”