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Effect of liquid-based cytology fixing solution on immunocytochemistry: Efficacy of antigen retrieval in cytologic
Nanako Sakabe1, Sayumi Maruyama1, Chihiro Ito1
1Pathophysiology Sciences, Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Insights
Immunocytochemistry (ICC) on liquid-based cytology (LBC) specimens shows improved results with antigen retrieval (AR). However, optimal staining for ICC depends on the specific antigen, cell type, and LBC fixing solution used.
Area of Science:
- Cytopathology
- Immunohistochemistry
- Diagnostic Pathology
Background:
- Immunocytochemistry (ICC) enhances diagnostic accuracy in cytology.
- Liquid-based cytology (LBC) is a common method for specimen preparation.
- Appropriate fixation is crucial for successful ICC on LBC specimens.
Purpose of the Study:
- To investigate the impact of LBC fixing solutions on ICC.
- To evaluate the efficacy of antigen retrieval (AR) in LBC specimens for ICC.
Main Methods:
- Utilized five types of LBC-fixed samples (SurePath™) with cell lines.
- Performed ICC using 13 antibodies.
- Analyzed results by quantifying positive cells in stained specimens.
Main Results:
- Heat-induced AR (HIAR) significantly improved ICC reactivity for nuclear antigens.
- Reactivity varied based on LBC fixing solutions (CytoRich™ Blue, CytoRich™ Red, TACAS™ Ruby) and specific antigens (e.g., Ki-67, estrogen receptor, p63, cytokeratin 5/6).
- Cytoplasmic and cell membrane antigens showed reduced reactivity without HIAR and with certain LBC fixatives.
Conclusions:
- ICC on LBC specimens is a valuable diagnostic tool.
- Staining conditions, including the choice of LBC fixing solution and the need for AR, must be optimized for each antigen.
- The interplay between antigen, cell type, and fixative impacts ICC immunoreactivity.
Background:
Immunocytochemistry (ICC) is an indispensable technique to improve diagnostic accuracy. ICC using liquid-based cytology (LBC)-fixed specimens has been reported. However, problems may arise if the samples are not fixed appropriately. We investigated the relationship between the LBC fixing solution and ICC and the usefulness of antigen retrieval (AR) in LBC specimens.
Methods:
Specimens were prepared from five types of LBC-fixed samples using cell lines and the SurePath™ method. ICC was performed using 13 antibodies and analyzed by counting the number of positive cells in the immunocytochemically stained specimens.
Results:
Insufficient reactivity was observed using ICC without heat-induced AR (HIAR) in nuclear antigens. The number of positive cells increased in ICC with HIAR. The percentage of positive cells was lower in CytoRich™ Blue samples for Ki-67 and in CytoRich™ Red and TACAS™ Ruby samples for estrogen receptor and p63 than in the other samples. For cytoplasmic antigens, the percentage of positive cells for no-HIAR treatment specimens was low in the three antibodies used. In cytokeratin 5/6, the number of positive cells increased in all LBC specimens with HIAR, and the percentage of positive cells in CytoRich™ Red and TACAS™ Ruby samples was significantly lower (p < .01). For cell membrane antigens, CytoRich™ Blue samples had a lower percentage of positive cells than the other LBC-fixed samples.
Conclusion:
The combination of detected antigen, used cells, and fixing solution may have different effects on immunoreactivity. ICC using LBC specimens is a useful technique, but the staining conditions should be examined before performing ICC.
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