Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH)

Stuart Schwartz1

1 Cytogenetics Laboratory, Laboratory Corporation of America® Holdings, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.9
DOI:  10.1002/0471142905.hg0809s85
Online Posting Date:  April, 2015
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FISH has been used to detect and clarify deletions and/or other structural rearrangements, and also has applications in interphase analysis. This unit describes preparation of uncultured amniotic fluid cells for FISH analysis. Cells are swollen, and then slides are prepared by standard methods. The cells are then fixed and permeabilized for subsequent FISH. An alternate protocol describes attachment of amniocytes to a glass or plastic surface followed by hypotonic swelling, fixation, and permeabilization for subsequent FISH. Interphase FISH analysis of amniotic fluid cells is also described. © 2015 by John Wiley & Sons, Inc.

Keywords: prenatal diagnosis; FISH; fluorescence in situ hybridization; amniotic fluid; aneuploidy

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Uncultured Amniocytes for Interphase FISH Analysis
  • Alternate Protocol 1: Preparation of Amniocytes Attached to a Surface for Interphase FISH Analysis
  • Basic Protocol 2: Interphase FISH Analysis of Amniotic Fluid Cells
  • Basic Protocol 3: Signal Enumeration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Uncultured Amniocytes for Interphase FISH Analysis

  • Whole amniotic fluid specimen
  • 1× trypsin/EDTA (0.05% trypsin/0.53 mM EDTA·4Na in Hanks’ Balanced Salt Solution without or Mg)
  • Hypotonic solution: 0.56% (w/v) KCl
  • Fixative: 3:1 (v/v) methanol:glacial acetic acid
  • 2× SSC, pH 5.3 ( appendix 2D)
  • Pepsin working solution: 2.5 mg pepsin added to 50 ml of 0.01 N HCl
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • Post‐fixation solution (see recipe)
  • 70%, 85%, and 100% ethanol (see recipe for ethanol wash solutions)
  • 15‐ml screw‐cap conical centrifuge tubes (e.g., BD Falcon)
  • Clinical tabletop centrifuge (e.g., IEC Centra‐HN)
  • Glass microscope slides, pre‐cooled
  • Cotton‐plugged Pasteur pipets
  • Additional reagents and equipment for chromosome slide preparation (unit 4.1; Bangs and Donlon, )

Alternate Protocol 1: Preparation of Amniocytes Attached to a Surface for Interphase FISH Analysis

  Additional Materials (also see protocol 1)
  • Chang in situ medium (Irvine Scientific) supplemented with penicillin/streptomycin (100 U/ml penicillin/100 μg/ml streptomycin added from 100× stock; BioWhittaker) or Amniomax complete medium (Life Technologies)
  • Additional reagents and equipment for in situ preparation, harvest, and culture of amniotic fluid samples (unit 8.4; Minehart Miron, )
  • NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Interphase FISH Analysis of Amniotic Fluid Cells

  • Denaturing solution (see recipe), 73°C70%, 85%, and 100% ethanol (see recipe for ethanol wash solutions), ice cold
  • Slide or coverslip containing amniotic fluid cells (see protocol 1 or see Alternate Protocol)
  • Probes (Vysis/Abbot):
    • CEP 18 (alpha‐satellite DNA probe)
    • CEP X (alpha‐satellite DNA probe)
    • CEP Y (alpha‐satellite DNA probe)
    • LSI 13
    • LSI 21
  • Rubber cement
  • 0.4× SSC/0.3% NP‐40 solution (see recipe)
  • 2× SSC/0.1% NP‐40 solution (see recipe)
  • 2 × SSC ( appendix 2D), 37°C
  • Moist chamber (unit 4.3) Coplin jars
  • pH meter or pH paper
  • 73°C water bath
  • Forceps
  • 45° to 50°C slide warmer
  • 22 × 22–mm glass coverslips
  • 5‐ml syringe
  • DAPI II counterstain
  • Fluorescence microscope with epi‐illumination and filter set appropriate for fluorochrome used [(unit 4.3 (Knoll and Lichter, ) and unit 4.4 (McNamara et al., )]
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Literature Cited

Literature Cited
  Bangs, C.D. and Donlon, T.A. 2005. Metaphase chromosome preparation from cultured peripheral blood cells. Curr. Protoc. Hum. Genet. 45:4.1.1‐4.1.19.
  Benn, P., Ciarleglio, L., Lettieri, L., Rosdis, J., and Greenstein, R. 1992. A rapid (but wrong) prenatal diagnosis. N. Engl. J. Med. 326:1638‐1640.
  Bubendorf, L. and Piaton, E. 2012; UroVysion® multiprobe FISH in the triage of equivocal urinary cytology cases. Ann. Pathol. Dec. 32:e52‐e56, 438‐443.
  Choy, R.K., Chen, Y., Sun, X.F., Kwok, Y.K., and Leung, T.Y. 2014. BACs‐on‐beads: A new robust and rapid detection method for prenatal diagnosis. Exp. Rev. Mol. Diagn. 14:273‐280.
  Das, K. and Tan, P. 2013. Molecular cytogenetics: Recent developments and applications in cancer. Clin. Genet. 84:315‐325.
  Faas, B.H., Cirigliano, V., and Bui, T.H. 2011. Rapid methods for targeted prenatal diagnosis of common chromosome aneuploidies. Semin. Fetal Neonatal Med. 16:81‐87.
  Haferlach, T. 2012. Molecular genetics in myelodysplastic syndromes. Leuk. Res. 36:1459‐1462.
  Jehan, Z., Uddin, S., and Al‐Kuraya, K.S. 2012. In‐situ hybridization as a molecular tool in cancer diagnosis and treatment. Curr. Med. Chem. 19:3730‐3738.
  Klinger, K., Landes, G., Shook, D., Harvey, R., Lopez, L., Locke, P., Lerner, T., Osathanondh, R., Leverone, B., Houseal, T., Pavelka, K., and Dackowski, W. 1992. Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH). Am. J. Hum. Genet. 51:55‐65.
  Knoll, J.H.M. and Lichter, P. 2005. In situ hybridization to metaphase chromosomes and interphase nuclei. Curr. Protoc. Hum. Genet. 45:4.3.1‐4.3.31.
  Lampert, F., Harbott, J., and Borkhardt, A. 2013. Cytogenetic aspects of childhood leukemias. Klin. Padiatr. 225:S30‐S33.
  McNamara, G., Difilippantonio, M.J., and Ried, T. 2005. Microscopy and image analysis. Curr. Protoc. Hum. Genet. 46:4.4.1‐4.4.34.
  Minehart Miron, P. 2012. Preparation, culture, and analysis of amniotic fluid samples. Curr. Protoc. Hum. Genet. 74:8.4.1‐8.4.14.
  Nub, S., Brebaum, D., and Grond‐Ginsbach, C. 1994. Maternal cell contamination in amniotic fluid samples as a consequence of the sampling technique. Hum. Genet. 93:121‐124.
  Rebello, M.T., Abas, A., Nicolaides, K., and Coleman, D.V. 1994. Maternal contamination of amniotic fluid demonstrated by DNA analysis. Prenat. Diag. 14:109‐112.
  Riegel, M. 2014. Human molecular cytogenetics: From cells to nucleotides. Genet. Mol. Biol. 37:194‐209.
  Rodríguez, L., Liehr, T., Mrasek, K., Mansilla, E., Martínez‐Fernández, M.L., Garcia, A., and Martínez‐Frías, M.L. 2007. Small supernumerary chromosome marker generating complete and pure trisomy 18p, characterized by molecular cytogenetic techniques and review. Am. J. Med. Genet. A 143A:2727‐2732.
  Stumm, M. and Tönnies, H. 2008. Fluorescence in situ hybridization techniques in medical diagnostics. Exp. Opin. Med. Diagn. 2:1381‐1390.
  Tepperberg, J., Pettenati, M.J., Rao, P.N., Lese, C.M., Rita, D., Wyandt, H., Gersen, S., White, B., and Schoonmaker, M.M. 2001. Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2‐year multi‐center retrospective study and review of the literature. Prenat. Diagn. 21:293‐301.
  Test and Technology Transfer Committee, American College of Medical Genetics. 2000. Technical and clinical assessment of fluorescence in situ hybridization: An ACMG/ASHG position statement. I. Technical considerations. Test and Technology Transfer Committee. Genet. Med. 2:356‐361.
  Tsuchiya, K.D. 2011. Fluorescence in situ hybridization. Clin. Lab. Med. 31:525‐542, vii‐viii.
  Ward, B.E., Gersen, S.L., Carelli, M.P., McGuire, N.M., Dackowski, W.R., Weinstein, M., Sandlin, C., Warren, R., and Klinger, K.W. 1993. Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: Clinical experience with 4,500 specimens. Am. J. Hum. Genet. 52:854‐865.
  Weise, A. and Liehr, T. 2008. Fluorescence in situ hybridization for prenatal screening of chromosomal aneuploidies. Exp. Rev. Mol. Diagn. 8:355‐357.
  Zuffardi, O, Bonaglia, M., Ciccone, R., and Giorda, R. 2009. Inverted duplications deletions: Underdiagnosed rearrangements? Clin. Genet. 75:505‐513.
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