Comparison of Fixation Methods for Preservation Cytology Specimens of Cell Block Preparation Using 10% Neutral Buffer Formalin and 96% Alcohol Fixation in E-cadherin and Ki-67 Immunohistochemical Examination

Abstract

BACKGROUND: Cytological and molecular examinations are among the most important examinations in cancer diagnosis. 96% alcohol is a fixative solution commonly used by clinicians for cytological samples because of its accessibility and affordability. Cellblock preparation from cytology specimen may increase morphology detail and may be used for further biomarker analysis. E-cadherin is an adhesion protein expressed in the cell membrane of most carcinoma. Ki67 is a protein expressed in nuclei of malignant cells that used as a proliferation marker.
 AIM: This study was designed to investigate the effect of fixation duration in 96% alcohol on protein preservation for immunohistochemistry (IHC) evaluation compared to 10% neutral buffered formalin (NBF) as the gold standard.
 METHODS: Twenty-five fine-needle aspiration biopsy (FNAB) specimen diagnosed as carcinoma were fixed in 10% NBF and 96% alcohol for 1 hour, 6 hours, 24 hours, 48 hours and 72 hours. Cell blocks preparation were made from those 6 groups of specimens. E-cadherin and Ki67 IHC were done to cell blocks section and evaluated. The data were statistically analysed using the Friedman test with p-value < 0.05 of a significant level.
 RESULTS: There were significant differences between E-cadherin and Ki67 expression in cell block preparation from 96% alcohol-fixed cytology specimen for 1 hour, 6 hours, 24 hours, 48 hours and 72 hours to 10% NBF (p = 0.0001).
 CONCLUSION: The result indicated that 96% alcohol is not suitable as a fixative solution for cell block preparation in E-cadherin and Ki-67 IHC examination.

Comparison of Fixation Methods for the Detection of Claudin 1 and E-Cadherin in Breast Cancer Cell Lines by Immunofluorescence

“…In other cell models, formaldehyde or alcohol-based fixatives (including methanol) have alternatively been used for the immunofluorescence detection of claudin 1 18,20,21 or E-cadherin. 22,23 Interestingly, the direct comparison of different fixing protocols including formaldehyde and methanol fixation for the detection of claudin 1 in airway epithelial cells concluded that methanol fixation was optimal to obtain consistent and reliable signals. 24 Altogether, these observations strongly suggest that the best fixation method depends on the sample type, cell type, or experimental conditions.…”

The Yokohama System for Reporting Breast Cytopathology

“…19,20 Cell block section is an ideal sample type to perform immunocytochemical study because the sample processing is similar to that of histologic section. [21][22][23] However, when cell block is not available, ER and PR can be tested on Papanicolaou-stained smear as long as in-house validation has been performed (Fig. 4).…”

Comparison of immunohistochemical mesothelial biomarkers in paired biopsies and effusion cytology cell blocks from pleural mesothelioma

“…12,13 It is known that differences in fixation and preparation of cytology compared to biopsies may affect the results of IHC. [14][15][16] Only a few studies have included a limited number of mesotheliomas with paired biopsies and effusion cytology specimens for comparison of protein expression by IHC. [17][18][19][20][21][22] In a previous study, we showed that IHC assessment of PD-L1 in paired specimens from pleural mesotheliomas is comparable, but we found a higher concordance between biopsies and effusions in epithelioid than in non-epithelioid mesotheliomas, 23 which may indicate biological differences.…”

“…It is known that differences in fixation and preparation of cytology compared to biopsies may affect the results of IHC 14–16 . Only a few studies have included a limited number of mesotheliomas with paired biopsies and effusion cytology specimens for comparison of protein expression by IHC 17–22 .…”