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Interrelation of formalin fixation, chromatin compactness and DNA values as measured by flow and image cytometry
1Department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000.
Abstract:
The severity and consistency of the effect of formalin fixation on the quantitation of DNA by flow cytometry (FCM) and image cytometry (ICM) were studied. As compared to ethanol, formalin fixation substantially decreased the propidium iodide fluorescence from mouse hepatocyte nuclei analyzed by FCM; it was also associated with an altered 4n-to-2n signal ratio and with false aneuploid peaks by FCM, but not by ICM (microspectrophotometry). ICM, on the other hand, suffered from a dependence of the DNA signal on nuclear size, which was not seen with FCM. The DNA signal variation was related to variations in the chromatin state, as shown by differences between monocytes and lymphocytes, and between RAJI cells fixed under various ionic strengths. The dependence of the DNA signal on the chromatin state indicates a need for caution in interpreting aneuploidy in formalin-fixed cells. For FCM, pseudoaneuploidy appears avoidable by using a Feulgen fluorescence staining technique. New imaging modes may be necessary to solve the problem of cell size dependence for ICM DNA determination.
Insights
Formalin fixation significantly impacts DNA quantitation accuracy in flow cytometry (FCM) and image cytometry (ICM). Formalin alters DNA signals, potentially causing false aneuploidy in FCM, while ICM shows cell size dependency, necessitating careful interpretation of DNA content in fixed cells.
Area of Science:
- Cellular Biology
- Biotechnology
- Cytometry
Background:
- Accurate DNA quantitation is crucial for cell cycle analysis and ploidy determination.
- Formalin fixation is a common method for preserving biological samples but can affect cellular components.
Purpose of the Study:
- To investigate the effects of formalin fixation on DNA quantitation using flow cytometry (FCM) and image cytometry (ICM).
- To compare formalin fixation with ethanol fixation regarding DNA measurement accuracy.
Main Methods:
- Mouse hepatocyte nuclei were analyzed using FCM and ICM after fixation with formalin or ethanol.
- Propidium iodide fluorescence and DNA signal variations were measured.
- Chromatin state variations were assessed by comparing different cell types and fixation conditions.
Main Results:
- Formalin fixation significantly reduced propidium iodide fluorescence in FCM compared to ethanol.
- Formalin fixation led to altered 4n-to-2n DNA signal ratios and false aneuploid peaks in FCM, but not ICM.
- ICM exhibited DNA signal dependence on nuclear size, a phenomenon not observed in FCM.
- DNA signal variations correlated with chromatin states, as evidenced by differences between monocytes and lymphocytes.
Conclusions:
- Formalin fixation introduces significant artifacts in DNA quantitation by FCM and ICM, requiring cautious interpretation of aneuploidy.
- Pseudoaneuploidy in FCM can be mitigated using Feulgen fluorescence staining.
- Advanced imaging techniques may be required to address cell size-dependent DNA measurement issues in ICM.