Flow cytometric karyotyping: from centromeres to locus specific identification

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.