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Combined Immunofluorescence and FISH: New Prospects for Tumor Cell Detection/Identification

Peter F. Ambros¹, Gábor Méhes²

¹Children's Cancer Research Institute (CCRI), St. Anna Children's Hospital, Vienna, Austria
²University of Pécs, Pécs, Hungary

Publication Name:

Current Protocols in Cytometry

Unit Number:

Unit 8.13

DOI:

10.1002/0471142956.cy0813s26

Online Posting Date:

November, 2004

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Abstract

Fluorescence-microscope based methods are presented that allow automatic selection and quantification of immunophenotyped cells and sequential FISH analysis to facilitate molecular cytogenetic analysis of single cells and very small cell populations. The protocols in this unit are particularly useful for studying the biological and genetic makeup of disseminated tumor cells or other rare cells.

Keywords: fluorescence microscopy; immunofluorescence labeling; FISH; cytogenetic analysis

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Unit Introduction
Basic Protocol 1: Immunofluorescence Detection and Characterization of Rare Cells
Basic Protocol 2: FISH After Immunofluorescence Staining
Alternate Protocol 1: Sequential FISH Using Directly Labeled DNA Probes Following Immunofluorescent Analysis
Alternate Protocol 2: Multiple Sequential FISH Following Immunolabeling
Support Protocol 1: Mononuclear Cell Preparation for Sequential Immunolabeling
Support Protocol 2: Automated Fluorescence Image Analysis
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Immunofluorescence Detection and Characterization of Rare Cells

Materials

Cytological specimen: cytospin preparation (see, e.g., Support Protocol 1), smear, or touch slide
3.7% (v/v) formaldehyde in PBS (see appendix 2A for PBS)
Phosphate-buffered saline (PBS), pH 7.0 (appendix 2A)
2% and 4% (w/v) bovine serum albumin (BSA; Sigma) in PBS (see appendix 2A); filter sterilize before use
Primary antibody: e.g., anti-cytokeratin (MNF 116, available from Dako) or anti-GD2 (available from Dr. R. Reisfeld, Scripps Clinic, La Jolla, Calif.)
Fluorescently labeled secondary antibody directed against species in which primary antibody was raised: e.g., FITC-conjugated anti-mouse
0.2 µg/ml DAPI (Sigma) in PBS (see appendix 2A)

Moist chamber: e.g., rectangular plastic box with lid, containing paper towels moistened with deionized H₂O, accommodating up to 10 slides
Glass coverslips
Antifade mounting medium containing DAPI (e.g., Vectashield, Vector Laboratories)
Fluorescence microscope (e.g., Axioplan, Zeiss)
Digital image-analysis workstation (optional; see Support Protocol 2)

Basic Protocol 2: FISH After Immunofluorescence Staining

Materials

Immunofluorescently labeled slides (see Basic Protocol 1)
Phosphate-buffered saline (PBS), pH 7.0 (appendix 2A)
0.05% pepsin solution (see recipe)
70%, 96%, and 100% ethanol
Hybridization mix (see recipe) containing biotin- or digoxigenin-labeled DNA probes (ideally BAC, PAC, YAC, or cosmid clones or repetitive sequences)
Rubber cement
2× and 4× SSC (appendix 2A)
Post-hybridization wash solution: e.g., 50% (v/v) formamide in 2× SSC, prewarmed to 42°C
2% and 4% (w/v) bovine serum albumin (BSA; Sigma) in PBS (see appendix 2A); filter sterilize before use
Primary antibodies: e.g., mouse anti-biotin (Dako), FITC-conjugated sheep anti-digoxigenin (e.g., Boehringer Mannheim)
Secondary antibodies: fluorochrome-conjugated antibodies directed against species from which primary antibody was derived (e.g., TRITC-conjugated rabbit anti-mouse, FITC-conjugated rabbit anti-sheep (e.g., Dako))
0.1% (v/v) Tween 20 in 4× SSC (see appendix 2A for SSC)
3.7% (v/v) formaldehyde in PBS (see appendix 2A for PBS)
Antifade mounting medium containing DAPI (e.g., Vectashield, Vector Laboratories)
Coplin jars
Moist chamber: e.g., rectangular plastic box with lid, containing paper towels moistened with deionized H₂O, accommodating up to 10 slides
Coverslips

Alternate Protocol 1: Sequential FISH Using Directly Labeled DNA Probes Following Immunofluorescent Analysis

Additional Materials (also see Basic Protocol 2)

DNA probes with direct fluorochrome labeling (purchase from Qbiogene, use LSI probes from Vysis, or prepare home-made probes as described in unit 8.3) ; store DNA probes at –20°C; store ready-to-use probe mixtures at 4°C

Alternate Protocol 2: Multiple Sequential FISH Following Immunolabeling

Additional Materials (also see Basic Protocol 2)

65% (v/v) formamide in 2× SSC (see appendix 2A for SSC)

Support Protocol 1: Mononuclear Cell Preparation for Sequential Immunolabeling

Materials

Lymphoprep (Nycomed)
Phosphate-buffered saline (PBS), pH 7.0 (appendix 2A)
Erythrolysis buffer (see recipe)
RPMI 1640 medium containing 10% FBS (appendix 3B)

240-mm² Hettich Cytospin cytocentrifuge
15-ml conical polypropylene tubes
Benchtop centrifuge
Glass slides (e.g., HistoBond from Paul Marienfeld, Heidelberg, Germany)
Vacuum aspirator

Additional reagents and equipment for counting cells using a Coulter counter (appendix 3A)

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Figures

Figure 8.13.1

Schematic presentation of the work flow of the automatic microscope.

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Figure 8.13.2

Gallery of automatically selected breast carcinoma cells showing double-positive immunofluorescence staining with cytokeratin (FITC) and mucin (TRITC), and DAPI-positive nuclei.

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Figure 8.13.3

(A) Breast carcinoma cell surrounded by hematopoietic cells visualized by cytokeratin-positive staining (FITC). (B) The tumor origin was verified by sequential FISH using a chromosome 17 (TRITC) and an X-specific (FITC) DNA probe.

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Figure 8.13.4

(A) Leukemia cell: positive staining with CD10 (TRITC) and proliferation-associated marker Ki-67 (FITC). (B) Leukemia-specific genetic aberrations: trisomy of chromosome 16 (TRITC) and monosomy X (FITC). (C) TEL/AML fusion (arrow). Aberrations and fusion were demonstrated sequentially on the same cell after automatic relocation of the immunologically positive cell, thus providing the ultimate proof of the leukemic nature of the target cell.

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Literature Cited

Literature Cited
	Ambros, P.F., Méhes, G., Hattinger, C.M., Ambros, I.M., Luegmayr, A., Ladenstein, R., and Gadner, H. 2001. Unequivocal identification of tumor cells in the bone marrow by combining immunological and genetic approaches: Functional and prognostic information. Leukemia 15:275-277.
	Ambros, P.F., Méhes, G., Ambros, I.M., and Ladenstein, R. 2003. Disseminated tumor cells in the bone marrow: Chances and consequences of microscopical detection methods. Cancer Lett. 197:29-34.
	Hopman, A.H., Voorter, C.E., and Ramaekers, F.C. 1994. Detection of genomic changes in cancer by in situ hybridization. Mol. Biol. Rep. 19:31-44.
	Knuutila, S. 1993. Simultaneous detection of immunophenotype and genome by the MAC method. J. Histochem. Cytochem. 41:1715-1716.
	Méhes, G., Lörch, T., and Ambros, P.F. 2000. Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis. Cytometry 42:357-362.
	Méhes, G., Luegmayr, A., Hattinger, C.M., Lörch, T., Ambros, I.M., Gadner, H., and Ambros, P.F. 2001. Combined automatic immunological and molecular cytogenetic analysis allows exact identification and quantification of tumor cells in the bone marrow. Clin. Cancer Res. 7:1969-1975.
	Nylund S.J., Wessman, M., and Larramendy, M.L. 1994. Analysis of genotype and phenotype on the same interphase or mitotic cell: A manual of MAC (morphology antibody chromosomes) methodology. Cancer Genet. Cytogenet. 72:1-15.
	Speel, E.J.M., Herbergs, J., Ramaekers, F.C.S., and Hopman, A.H.N. 1994. Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic and phenotypic parameters of tumor cells. J. Histochem. Cytochem. 42:961-966.
	Strehl, S. and Ambros, P.F. 1993. Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma. Cytogenet. Cell Genet. 63:24-28.
	Weber-Matthiesen, K., Deerberg, J., Müller-Hermelink, A., Schlegelberger, B., and Grote, W. 1993. Rapid immunophenotypic characterization of chromosomally aberrant cells by the new FICTION method. Cytogenet. Cell Genet. 63:123-125
The help of Andrea Luegmayr, Elisabeth Vitasek, and Rita Narath is greatly acknowledged. This work was supported by the Children's Cancer Research Institute (CCRI).