Preparation, Culture, and Analysis of Amniotic Fluid Samples

Patricia Minehart Miron1

1 UMass Memorial Medical Center, Worcester, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.4
DOI:  10.1002/0471142905.hg0804s74
Online Posting Date:  July, 2012
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Abstract

Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid is used directly for biochemical analyses and as a source of fetal cells for biochemical assays, DNA testing, and chromosome studies. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for chromosome analysis. Cells are grown, harvested for metaphase spreads, and analyzed on glass coverslips. The unit also describes methods to obtain cells for additional studies (such as molecular genetic analyses) by growing cells in flasks either following passaging of cells from a glass coverslip culture or by directly establishing a flask culture from the amniotic fluid specimen. When cells are grown in flasks, they must be removed from the flask with trypsin before they can be used in studies. Curr. Protoc. Hum. Genet. 74:8.4.1‐8.4.14. © 2012 by John Wiley & Sons, Inc.

Keywords: cytogenetics; amniotic fluid; clinical; metaphase chromosomes; prenatal diagnosis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Situ Method for the Preparation, Culture, Harvest, and Analysis of Amniotic Fluid Samples
  • Support Protocol 1: Passaging Cells from Coverslips
  • Support Protocol 2: Flask Method for Culturing Cells Directly from Amniotic Fluid Samples as a Source for Additional Studies
  • Support Protocol 3: Passaging Cells from Flasks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Situ Method for the Preparation, Culture, Harvest, and Analysis of Amniotic Fluid Samples

  Materials
  • 10 to 20 ml amniotic fluid
  • AmnioMax II Complete Medium see recipe), room temperature and 37°C
  • Fetal bovine serum (Invitrogen, cat. no. 12140079)
  • 10 mg/ml Colcemid (e.g., Invitrogen)
  • Hypotonic solution: 0.075 M KCl or 0.7% (w/v) sodium citrate
  • 6:1 and 3:1 (v/v) methanol/glacial acetic acid fixative (use ACS‐grade reagents; prepare fresh daily)
  • Permount
  • 20‐ml syringes or tubes approved for cell culture
  • 15‐ml centrifuge tubes
  • Centrifuge (e.g., Fisher Marathon 21K with four‐place rotor or Hettich Rotofix 32A)
  • 35‐mm tissue culture dishes containing coverslips (MatTek, cat. no. GSK/F)
  • 9‐in. (23‐cm) Pasteur pipet, autoclaved
  • Vacuum flask and vacuum source
  • Inverted microscope
  • Fine forceps
  • Blue pads or paper towels
  • 60°C slide warmer
  • Red china pencil or Gram stain pen
  • Coplin jars for coverslips
  • 3‐in. × 1‐in. × 1‐mm microscope slides (Curtin Matheson)
  • Additional reagents and equipment for cell culture ( appendix 3G), chromosome staining procedures including GTG banding (unit 4.2), and karyotyping ( appendix 4A)
CAUTION: Human amniotic fluid is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Passaging Cells from Coverslips

  • Cell cultures ready to be passaged ( protocol 1)
  • HBSS ( appendix 2D) without calcium and magnesium, 37°C
  • Trypsin/EDTA solution: 0.05% (w/v) trypsin/0.53 mM EDTA·4Na (Invitrogen), 37°C
CAUTION: Human cell cultures are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Flask Method for Culturing Cells Directly from Amniotic Fluid Samples as a Source for Additional Studies

  • Trypsin/EDTA solution: 0.05% (w/v) trypsin/0.53 mM EDTA·4Na (Invitrogen), 37°C
  • 25‐cm2 tissue culture flasks (Falcon; Fisher)
CAUTION: Human amniotic fluid is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 3: Passaging Cells from Flasks

  • Confluent or near‐confluent 25‐cm2 flask of cells ( protocol 3)
  • Trypsin/EDTA solution: 0.05% (w/v) trypsin/0.53 mM EDTA·4Na (Invitrogen), 37°C
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Figures

  •   FigureFigure 8.4.1 Mitotic refractory doublets in cultured cells from amniotic fluid, as viewed under an inverted microscope. Examples are indicated by the arrows.
  •   FigureFigure 8.4.2 Coverslip setups for cultured cells from amniotic fluid. (A) Leaning against top of a 35‐mm plate while drying (cell‐side‐up). (B) Two coverslips (cell‐side‐down) mounted on one microscope slide.
  •   FigureFigure 8.4.3 Flow chart of analysis of amniotic fluid samples. In this context, abnormal metaphases are those with (A) trisomies of chromosomes 8, 9, 13, 18, and 21; (B) sex chromosome aneuploidy; (C) marker chromosomes; and (D) unbalanced structural rearrangements. Adapted with permission from Frederick R. Bieber, Brigham and Women's Hospital, Boston.

Videos

Literature Cited

   Hsu, L.Y. and Benn, P.A. 1999. Revised guidelines for the diagnosis of mosaicism in amniocytes. Prenat. Diagn. 19:1081‐1090.
   Hsu, L.Y.F., Keefe, S., Jenkins, E.C., Alonso, L., Benn, P.A., David, K., Hirschorn, K., Lieber, E., Shanske, A., Shapiro, L.R., Schutta, E., and Warburton, D. 1992. Proposed guidelines for diagnosis of chromosome mosaicism in amniocytes based on data derived from chromosome mosaicism and pseudomosaicism studies. Prenatal Diag. 12:555‐573.
   Richkind, K.E. and Risch, N.J. 1990. Sensitivity of chromosomal mosaicism detection by different tissue culture methods. Prenatal Diag. 10:519‐527.
Key Reference
   Barch, M.J., Knutsen, T., and Spurbeck, JL (ed.) 1997. The AGT Cytogenetics Laboratory Manual, 3rd ed. The Association of Genetic Technologists, Lippincott‐Raven Publishers, New York.
  Provides a detailed overview of amniotic fluid cell culture and prenatal chromosome diagnosis (Chapter 5), and several protocols for the workup of amniotic fluid specimens (Chapter 6).
Internet Resources
   http://www.cap.org
  Cytogenetics checklist, College of American Pathologists.
   http://www.acmg.net
  Laboratory Standards and Guidelines, American College of Medical Genetics
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