Efficacy of the Antigenicity-Retaining Ability of Fixative Solutions for Liquid-Based Cytology: Immunocytochemistry of Long-Term Storage

Abstract

<b><i>Introduction/Objective:</i></b> Liquid-based cytology (LBC) is advantageous as multiple stained specimens can be prepared and used for additional assays such as immunocytochemical and molecular-pathological investigations. Two types of preservative-fixative solutions (fixatives) are used for nongynecologic specimens used in the BD SurePath-LBC (SP-LBC) method, and their components vary. However, few studies have evaluated the differences in antigen-retaining ability between these fixatives. Therefore, we investigated and compared the antigen-retaining ability of the fixatives in immunocytochemical staining (ICC) under long-term storage conditions. <b><i>Materials and Methods:</i></b> Sediments of cultured RAJI cells (derived from Burkitt’s lymphoma) were added to each fixative (red and blue) and stored at room temperature for a specified period (1 h; 1 week; and 1, 3, and 6 months). The specimens were then prepared using the SP-LBC method and subjected to ICC. Positivity rate was calculated using the specimens fixed at room temperature for 1 h as a control. Antibodies against Ki67 expressed in the nucleus and against CD20 and leukocyte common antigen (LCA) expressed on the cell membrane were used. <b><i>Results:</i></b> For CD20 and LCA, the positivity rate increased with time in the red fixative compared with that in the control. In the blue fixative, the positivity rate was highest at 1 h and was maintained at a high level throughout the storage period. In contrast, the Ki67 positivity rate was highest at 1 h in both red and blue fixatives and markedly decreased with time. Therefore, although refrigerated (8°C) storage was used, no improvement was noted. <b><i>Conclusions:</i></b> Long-term storage is possible for cell membrane antigens at room temperature; however, it is unsuitable for intranuclear antigens. Therefore, we conclude that suitable fixative type and storage temperature differ based on antigen location. Further investigation is warranted.

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

“…In particular, the LBC-fixed samples exhibited a significantly lower percentage of positive cells than the NBF-fixed sample for the nuclear antigen Ki-67 ( p < 0.01). This result may be due to alcohol, the main component of the four LBC fixing solutions, because the soluble proteins in the cytoplasm and nucleus are eluted during the dehydration and defatting processes [9, 19, 20]. Aldehyde fixation is suitable for detecting soluble proteins; however, the immunoreactivity of the Red and TAC samples was low.…”

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

“…[4][5][6][7] LBC is performed not only in the Papanicolaou test, but also in non-gynecologic cytology, 4 and many studies on ICC using LBC-fixed specimens have been reported. 12,16,17 Previously, we compared the effects of immunoreactivity between four types of LBC-and formalin-fixed samples on ICC. 20 To exclude the differences in the presence or absence of AR and permeability of antibody reagents due to differences in preparation methods and to reveal the effect of LBC fixing solutions on immunostaining, we performed ICC using CB specimens prepared from LBC-fixed samples post-fixed with formalin.…”

“…It has become an indispensable technique to improve the diagnostic accuracy of cytological diagnosis 4–7 . LBC is performed not only in the Papanicolaou test, but also in non‐gynecologic cytology, 4 and many studies on ICC using LBC‐fixed specimens have been reported 12,16,17 . Previously, we compared the effects of immunoreactivity between four types of LBC‐ and formalin‐fixed samples on ICC 20 .…”

“…Recently, studies have reported ICC using liquid-based cytology (LBC)-fixed specimens, which can be used to prepare multiple specimens of the same quality. 4,12,16,17 However, the standardization of ICC is challenging because many LBC fixing solutions with different ingredients and compositions are sold and used in cytology. The comparison of ICC between cell block (CB) specimens from LBC-fixed samples and FFPE sections may not yield consistent results, and ICC protocols need to be optimized prior to use in clinical practice.…”

“…In ICC, antigen retention and localization are important, and the antigen‐retaining ability of the fixing solution has a significant effect on the immunostaining results. Recently, studies have reported ICC using liquid‐based cytology (LBC)‐fixed specimens, which can be used to prepare multiple specimens of the same quality 4,12,16,17 . However, the standardization of ICC is challenging because many LBC fixing solutions with different ingredients and compositions are sold and used in cytology.…”