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Immunocytochemical Reactivity Comparison between Formalin-Fixed and Liquid-Based Cytology-Fixed Specimens
Nanako Sakabe1, Sayumi Maruyama2, Chihiro Ito2
1Pathophysiology Sciences, Department of Integrated Health Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan, nanako@met.nagoya-u.ac.jp.
Insights
Liquid-based cytology (LBC) fixing solutions can impact immunocytochemistry (ICC) results. Careful selection of LBC methods and antibodies is crucial for accurate ICC detection in cell block specimens.
Area of Science:
- Cytopathology
- Immunohistochemistry
- Biomedical research
Background:
- Liquid-based cytology (LBC) offers advantages for preparing multiple specimens and performing immunocytochemistry (ICC).
- However, LBC fixing solutions can alter antigenicity and affect ICC results.
- This study investigates the impact of different LBC fixing solutions on ICC immunoreactivity.
Purpose of the Study:
- To evaluate the effect of various LBC fixing solutions on the immunoreactivity of different antigens.
- To compare ICC results from LBC-fixed samples with those from traditional 10% neutral buffered formalin (NBF)-fixed samples.
Main Methods:
- Cell line samples were prepared using four LBC fixing solutions (PreservCyt®, CytoRich™ Red, CytoRich™ Blue, TACAS™ Ruby) and NBF fixation.
- Specimens were post-fixed with NBF for ICC analysis.
- Immunocytochemical staining was performed using 24 different antibodies, and the percentage of positive cells was quantified.
Main Results:
- Immunoreactivity varied based on antigen type and fixing solution.
- Nuclear antigens showed optimal results with NBF or TACAS™ Ruby, while cytoplasmic antigens performed well or better in LBC-fixed samples.
- Significant reductions in positive cell percentages were observed for CEA, Ber-EP4, CD10, and D2-40 in specific LBC formulations compared to NBF.
Conclusions:
- ICC on NBF-post-fixed LBC cell block specimens can yield comparable immunoreactivity to NBF-fixed samples.
- The choice of LBC fixing solution influences antigen detection rates, depending on the antibody and antigen abundance.
- Careful consideration of LBC methods and antibody selection is recommended for reliable ICC results.
Introduction:
Liquid-based cytology (LBC)-fixed samples can be used for preparing multiple specimens of the same quality and for immunocytochemistry (ICC); however, LBC fixing solutions affect immunoreactivity. Therefore, in this study, we examined the effect of LBC fixing solutions on immunoreactivity.
Methods:
Samples were cell lines, and specimens were prepared from cell blocks of 10% neutral buffered formalin (NBF)-fixed samples and the four types of LBC-fixed samples: PreservCyt®, CytoRich™ Red, CytoRich™ Blue, and TACAS™ Ruby, which were post-fixed with NBF. ICC was performed using 24 different antibodies, and immunocytochemically stained specimens were analyzed for the percentage of positive cells.
Results:
Immunoreactivity differed according to the type of antigen detected. For nuclear antigens, the highest percentage of positive cells of Ki-67, WT-1, ER, and p63 was observed in the NBF-fixed samples, and the highest percentage of positive cells of p53, TTF-1, and PgR was observed in the TACAS™ Ruby samples. For cytoplasmic antigens, the percentage of positive cells of CK5/6, Vimentin, and IMP3 in LBC-fixed samples was higher than or similar to that in NBF-fixed samples. The percentage of positive cells of CEA was significantly lower in CytoRich™ Red and CytoRich™ Blue samples than in the NBF-fixed sample (p < 0.01). Among the cell membrane antigens, the percentage of positive cells of Ber-EP4, CD10, and D2-40 was the highest in NBF-fixed samples and significantly lower in CytoRich™ Red and CytoRich™ Blue samples than that in NBF-fixed samples (p < 0.01). The NBF-fixed and LBC-fixed samples showed no significant differences in the percentage of positive cells of CA125 and EMA.
Discussion/Conclusion:
ICC using LBC-fixed samples showed the same immunoreactivity as NBF-fixed samples when performed on cell block specimens post-fixed with NBF. The percentage of positive cells increased or decreased based on the type of fixing solution depending on the amount of antigen in the cells. Further, the detection rate of ICC with LBC-fixed samples varied according to the type of antibody and the amount of antigen in the cells. Therefore, we propose that ICC using LBC-fixed samples, including detection methods, should be carefully performed.
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