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Robust 3D DNA FISH Using Directly Labeled Probes

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Published on: August 15, 2013

Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation.

Huey-En Tzeng¹, Yi-Wei Lee², Chien-Ting Lin³

¹Division of Hematology/Medical Oncology, Department of Medicine, Taichung Veterans General Hospital, Taichung City, Taiwan; Ph.D. Program for Cancer Molecular Biology and Drug Discovery, and Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; Department of Post-Baccalaureate Medicine, College of Medicine, National Chung-Hsing University, Taichung, Taiwan.

|June 9, 2024

Summary

This study introduces a new multicolour flow cytometry method for detecting nuclear FISH signals and cell surface markers simultaneously. This advance enables precise chimerism analysis and trisomy 12 detection in clinical samples.

Keywords:

FISH Flow-FISH cell immunophenotyping flow cytometry lineage-specific FISH

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Published on: February 6, 2012

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Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

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Published on: February 6, 2012

Area of Science:

Cytometry and Genetics
Molecular Biology
Clinical Diagnostics

Background:

Interphase chromosome (IC) Flow-FISH protocols are limited to single-color detection.
DNA denaturation in FISH disrupts cellular integrity, hindering concurrent surface marker analysis.
Existing methods struggle to combine multicolour FISH with flow cytometry for comprehensive cellular analysis.

Purpose of the Study:

To develop a protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers.
To validate the protocol for chimerism status in post-hematopoietic stem cell transplant patients.
To adapt the method for detecting trisomy 12 in chronic lymphocytic leukemia (CLL).

Main Methods:

Developed a novel protocol for multicolour IC Flow-FISH combined with surface marker labeling.
Validated the protocol using sex chromosome analysis for chimerism in blood cells.
Applied the method to detect trisomy 12 in CLL patient samples.

Main Results:

Demonstrated high agreement in detecting multiple colours and concurrent nuclear/surface signals.
Achieved high correlation (R² > 0.96) with conventional FISH for chimerism analysis in 56 clinical samples.
Successfully differentiated trisomy 12 signal localization in B cells vs. T cells and detected rare residual CLL cells post-transplant.

Conclusions:

The adaptable multicolour, lineage-specific IC Flow-FISH protocol overcomes previous limitations.
This method offers potential for various clinical applications currently using conventional FISH.
The technique enhances diagnostic capabilities for chromosomal aberrations and chimerism analysis.