Flow cytometric karyotyping: from centromeres to locus specific identification

Cuc Do, Karen Lower, Cindy Macardle, Bryone Kuss

    Research output: Contribution to journalMeeting Abstract

    Abstract

    Abstract Details: It is clear that 17p deleted clones
    represent high risk disease elements. However, the
    importance and the ability to accurately identify the
    percentages of 17p-deleted cells at diagnosis and during
    disease progression in chronic lymphocytic leukemia
    (CLL) remain unclear. Current methods to detect 17pdeleted
    cells, including microscopy-based fluorescence
    in situ hybridization (FISH) and full karyotyping, have
    restrictions on their lower limit of detection due to the low number of cells targeted. Additionally, characterising
    the immunophenotypic profiles of these early and
    often small sub-clones has not been possible. We have
    explored a novel technology: fluorescence in situ
    hybridization in suspension (FISH-IS) which incorporates
    a flow cytometry-based imaging approach with automated
    analysis of thousands of cells by Image Stream X.
    The initial aim of this study has validated the FISH-IS
    workflow with centromere probes (centromere Y, X, 9,
    12) on CLL samples to test the sensitivity and accuracy in
    distinguishing aneuploidy sub-groups (Figure 1).
    Secondly, we have investigated the FISH-IS assay with
    17p locus-specific probes on CLL samples. We have
    attempted this FISH-IS workflow using labelled bacterial
    artificial chromosome (BAC) DNA from RP11 library,
    using multiple labelled BAC probes to amplify the
    fluorescence signal. Background fluorescence has been a
    significant technical challenge. However, using 17p BAC
    probes optimised for label incorporation for maximum
    signal intensity and column based size selection to
    remove potential non-specific small probes, both signal
    intensity and background fluorescence are being
    addressed. A range of technical approaches has been applied and is presented. Flow sorting on the basis of
    FISH probe intensity is being explored. The optimisation
    of this method would enable its use as a prognostic test
    to detect very low frequency 17p deleted clones with a
    reliable number of cells monitored, potentially guiding
    clinicians to select the most appropriate therapy regimes
    for patients with a low-level 17p deletion at diagnosis.
    Importantly, the low frequency 17p deleted cells can
    potentially be flow sorted to facilitate sub-clone analysis,
    enabling questions on drug resistance mechanisms to
    be further explored.
    Original languageEnglish
    Article numberAbstract 79
    Pages (from-to)60-61
    Number of pages3
    JournalLeukemia and Lymphoma
    Volume56
    Issue numberS1
    DOIs
    Publication statusPublished - 19 Jan 2016
    EventXVI International Workshop on Chronic Lymphocytic Leukemia 2015 - Sydney, Australia
    Duration: 6 Sep 20159 Sep 2015

    Keywords

    • lymphocytic leukemia
    • cytometric katyotyping
    • locus specific identification

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