Immunocytochemical Reactivity Comparison between Formalin-Fixed and Liquid-Based Cytology-Fixed Specimens

Abstract

<b><i>Introduction:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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 (<i>p</i> < 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 (<i>p</i> < 0.01). The NBF-fixed and LBC-fixed samples showed no significant differences in the percentage of positive cells of CA125 and EMA. <b><i>Discussion/Conclusion:</i></b> 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.

Immunocytochemical detection by a fully automated immunostainer for assisting subclassification of pulmonary tumors on ThinPrep slides in real‐life clinical samples: A single‐institution experience

“…found that the percentage of positive cells increased or decreased based on the type of fixing solution depending on the amount of antigen in the cells of cell lines. 37 Although several results contrast with those of the present study, the cause of the variations in staining between investigations is unclear.…”

“…A study from a northern New England hospital showed concordant immunoreactivity for p63, TTF‐1, CD56, and Syn in CytoLyt‐fixed samples compared with FFPE material 35 . Sakabe et al found that the percentage of positive cells increased or decreased based on the type of fixing solution depending on the amount of antigen in the cells of cell lines 37 . Although several results contrast with those of the present study, the cause of the variations in staining between investigations is unclear.…”

Effect of liquid‐based cytology fixing solution on immunocytochemistry: Efficacy of antigen retrieval in cytologic specimens

“…Figure 4 shows the comparison of the positive rates between LBC specimens in this study and the previously reported CB specimens 20 in CK5/6 and D2‐40. In CK5/6, LBC specimens without HIAR had a lower positive rate than the CB specimens.…”

“…14 It is considered that AR is not necessary for Ber-EP4, MOC-31, EMA, and D2-40 examined in this study, because they have a higher immunoreactivity than previously reported CB specimens with HIAR. 20 In terms of the relationship between the immunoreactivity of cell membrane antigens and LBC fixing solutions, the positive rate of Blue samples was low in all five antigens examined. This result was similar to that of a previous study, 20 and some components in Blue may affect the antigenicity of the cell surface.…”

“…Comparison of percentage of positive cells for (A) CK5/6 and (B) D2‐40. Previous data 20 from CB specimens with HIAR were obtained by performing ICC on specimens prepared from LBC‐fixed samples post‐fixed with formalin. In CK5/6, the percentage of positive cells of LBC specimens without heat‐induced antigen retrieval (HIAR) was lower than that of CB specimens.…”