Preparation, Culture, and Analysis of Amniotic Fluid Samples
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
Table of Contents
- 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
- Literature Cited
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
Figure 8.4.1 Mitotic refractory doublets in cultured cells from amniotic fluid, as viewed under an inverted microscope. Examples are indicated by the arrows.
Figure 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.
|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.|
|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).|
|Cytogenetics checklist, College of American Pathologists.|
|Laboratory Standards and Guidelines, American College of Medical Genetics|