Analysis of Fine‐Needle Aspirate Biopsies from Solid Tumors by Laser Scanning Cytometry (LSC)

Andreas O.H. Gerstner1, Attila Tàrnok1

1 University of Leipzig, Leipzig, Germany
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.20
DOI:  10.1002/0471142956.cy0720s22
Online Posting Date:  November, 2002
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Abstract

Both flow and image cytometry have routine, reliable methods for ploidy analysis, each with its dbacks. LSC offers a non‐destructive and non‐consumptive method of analysis that allows repeated sample analysis as well as storage for later re‐evaluation. Owing to the extremely small sample requirement, specimens can be obtained through minimally invasive procedures such as fine‐needle aspirate biopsies or simple swabs. Tumors located at sites unsuitable for routine biopsy can be analyzed even on an out‐patient basis.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Ploidy in Carcinomas by LSC of Fine‐Needle Aspirate Biopsies
  • Alternate Protocol 1: Determination of Ploidy in Carcinomas by LSC of Eosinophil‐Rich Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of Ploidy in Carcinomas by LSC of Fine‐Needle Aspirate Biopsies

  Materials
  • Subject with solid tumor of interest
  • PBS with 1% and 0.5% BSA (see recipe)
  • Erythrocyte lysing solution: ammonium chloride lysing solution ( appendix 2A) or commercial lysing product (e.g., FACS Lysing Solution from Becton‐Dickinson)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • FITC‐conjugated anti‐cytokeratin antibody and negative‐control isotype (both mouse lgGs; Dako)
  • Anti‐FITC antibody conjugated to Alexa Green (Molecular Probes)
  • PBS with 0.5% BSA, 50 µg/ml PI, and 100 µg/ml RNase (see recipe)
  • Glycerol/PBS/PI (see recipe)
  • Rubber cement (optional)
  • Hematoxylin and eosin: purchase commercial H&E staining kit or see Zeller and Rogers ( )
  • Ethanol dehydration series: 70%, 80%, 90%, and 100% ethanol
  • Xylene
  • Permanent medium for cytological slides (e.g., Eukitt from Kindler GmbH)
  • 20‐ml syringe with 27‐G (or narrower) needle
  • Glass microscope slides, cover glasses, and grease pencil for use on glass slides
  • Humidified chamber
  • Laser scanning cytometer (CompuCyte) equipped with argon and helium‐neon lasers and standard filter settings
  • Additional reagents and equipment for hematoxylin/eosin staining (Zeller and Rogers, )

Alternate Protocol 1: Determination of Ploidy in Carcinomas by LSC of Eosinophil‐Rich Samples

  • Anti‐cytokeratin antibody and negative‐control isotype, both pure unconjugated IgG1 from mouse (Dako)
  • Anti‐mouse IgG antibody conjugated to biotin (Caltag)
  • Streptavidin conjugated to allophycocyanin (APC) (Caltag)
Replace steps and of the protocol 1 with steps 6a, 7a and 9a below.
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Figures

Videos

Literature Cited

Literature Cited
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   Franzén, G., Klausen, O.G., Grenko, R.T., Carstensen, J., and Nordenskjöld, B. 1991. Adenoid cystic carcinoma: DNA as an prognostic indicator. Laryngoscope 101:669‐673.
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   Gerstner, A.O.H., Machlitt, J., Welkoborsky, H.‐J., Bootz, F., Tárnok, A. 2002b. Analysis of ploidy in hypopharyngeal cancer by laser scanning cytometry on fine needle aspirate biopsies. Anal. Cell Pathol. In press
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   Kamentsky, L.A. and Kamentsky, L.D. 1991. Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data. Cytometry 12:381‐387.
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   Mighell, A.J. and High, A.S. 1998. Histological identification of carcinoma in 21 gauge needle tracks after fine needle aspiration biopsy of head and neck carcinoma. J. Clin. Pathol. 51:241‐243.
   Millot, C. and Dufer, J. 2000. Clinical applications of image cytometry to human tumour analysis. Histol. Histopathol. 15:1185‐1200.
   Sen, S. 2000. Aneuploidy and cancer. Curr. Opin. Oncol. 12:82‐88.
   Smith, E.H. 1991. Complications of percutaneous abdominal fine‐needle biopsy. Radiology 178:253‐258.
   Tárnok, A. and Gerstner, A.O.H. 2002. Clinical applications of laser scanning cytometry (LSC). Cytometry 50:133‐143.
   Welkoborsky, H.J., Gluckmann, J.L., Jacob, R., Bernauer, H., and Mann, W. 1999. Tumor biologic prognostic parameters in T1N0M0 squamous cell carcinoma of the oral cavity. Laryngorhinootologie 78:131‐138.
   Zeller, R. and Rogers, M. 1993. Counterstaining and mounting of autoradiographed in situ hybridization slides. In Current Protocols in Molecular Biology (F.A., Ausubel, R., Brent, R.E., Kingston, D.D. Moore, J.G., Seidman, J.A., Smith, and K. Struhl, eds.) pp. 14.5.12D14.5.5. John Wiley & Sons, New York.
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