Comparison of fixation procedures for fluorescent quantitation of DNA content using image cytometry.

Author(s): Maciorowski Z, Veilleux C, Gibaud A, Bourgeois CA, Klijanienko J, Boenders J, Vielh P

Publication: Cytometry, 1997, Vol. 28, Page 123-9

PubMed ID: 9181301 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of fixative and slide preparation method on fluorescence image and flow cytometry analysis of DNA in breast tumor cells and peripheral blood lymphocytes. After preparation, slides intended for fluorescent microscopy were stored at -20 degrees C prior to 4',6-diamidino-2-phenylindole (DAPI) staining and analysis. Fixed cell suspensions intended for flow cytometry were held at 4 degrees C overnight prior to analysis, while unfixed cells were analyzed within 30 minutes.

   Summary of Findings:

   The best image analysis histograms of both lymphocytes and fine needle aspirates of breast tumor cells were generated using paraformaldehyde-ethanol-fixed cells with slide preparation by cytocentrifugation or using ethanol-acetic acid-fixed cells and preparing slides by the standard air-dried drop method. For breast tumor cells, the fewest cell clumps and aggregates were seen when cells were fixed and slides were prepared by either of these two methods. Visualization of nuclear morphology was best when fixation of lymphocytes and breast tumor cells was with either ethanol or ethanol-acetic acid and breast tumor slides were prepared by cytocentrifugation rather than by the drop method. All methods of fixation yielded adequate flow cytometry results for both lymphocytes and breast tumor cells except for fixation with ethanol-acetic acid. Image analysis-calculated DNA indexes of aneuploid populations were 7-15% higher than those calculated by flow cytometry. The authors conclude that paraformaldehyde-ethanol fixation is useful in combination with presorting by flow cytometry while ethanol-acetic acid fixation is not as compatible with presorting by flow cytometry.

   Biospecimens

   • Cell - Breast
   • Cell - White Cells

   Preservative Types

   • Ethanol
   • None (Fresh)
   • Other Preservative

   Diagnoses:

   • Not specified
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Flow cytometry
   Cell count/volume	Flow cytometry
   DNA	Fluorescent microscopy
   Morphology	Fluorescent microscopy
   Cell count/volume	Fluorescent microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Ethanol Ethanol-acetic acid None (fresh) Paraformaldehyde-ethanol
   Biospecimen Aliquots and Components	Type of slide	Drop preparation Cytospin preparation
   Biospecimen Acquisition	Biospecimen location	Peripheral blood lymphocytes Breast cells
   Flow cytometry Specific	Technology platform	Image cytometry

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