Preparation of DNA from cytological material

Effects of Fixation, Staining, and Mounting Medium on DNA Yield and Quality

Authors

  • Annika Dejmek MD, PhD,

    1. Department of Clinical Pathology, University and Regional Laboratories Region Skåne, Malmo, Sweden
    2. Department of Laboratory Medicine, Center for Molecular Pathology, Lund University, Lund, Sweden
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  • Nooreldin Zendehrokh PhD,

    1. Department of Clinical Pathology, University and Regional Laboratories Region Skåne, Malmo, Sweden
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  • Malgorzata Tomaszewska MSc,

    1. Department of Clinical Chemistry, University and Regional Laboratories Region Skåne, Malmö, Sweden
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  • Anders Edsjö MD, PhD

    1. Department of Clinical Pathology, University and Regional Laboratories Region Skåne, Malmo, Sweden
    2. Department of Laboratory Medicine, Center for Molecular Pathology, Lund University, Lund, Sweden
    3. Department of Clinical Pathology and Cytology, Sahlgrenska University Hospital, Gothenburg, Sweden
    4. Department of Pathology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
    Current affiliation:
    1. Department of Clinical Pathology and Cytology, Sahlgrenska University Hospital, Gothenburg, Sweden
    2. Department of Pathology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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Corresponding author: Anders Edsjö, MD, PhD, Department of Clinical Pathology and Cytology, Sahlgrenska University Hospital, Gula Stråket 8, 413 45 Gothenburg, Sweden; Fax: (011) 46 (0)31 41 72 83; anders.edsjo@vgregion.se

Abstract

BACKGROUND

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material.

METHODS

DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol.

RESULTS

All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples.

CONCLUSIONS

Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344–353. © 2013 American Cancer Society.

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