CytoLyt® Fixation and Decalcification Pretreatments Alter Antigenicity in Normal Tissues Compared With Standard Formalin Fixation

Abstract

Immunohistochemistry is used on cell blocks constructed from cytopathology samples fixed in methanol-based fixatives, such as CytoLyt (Cytyc Corp), and on surgical pathology tissues exposed to decalcifying agents, often without technical validation. We evaluated a panel of commonly utilized antibodies in normal tissues exposed to differing preanalytic conditions as follows: CytoLyt fixation, formalin fixation followed by exposure to decalcifying agents (Leica Decalcifier I-10% formic acid or Leica Decalcifier II-5% hydrochloric acid), or standard formalin fixation. Altered expression was observed with several antibodies compared with standard formalin fixation. Specifically, there was absent or near absent expression of thyroid transcription factor 1 (TTF-1), D2-40, and CD20 in CytoLyt-fixed tissues, whereas reduced expression was observed for p63, estrogen receptor, S100 protein, CD3, calretinin, chromogranin, and synaptophysin. Absent or near absent expression of TTF-1 was also observed with exposure to hydrochloric acid, whereas reduced expression was observed for CK5/6, CK7, p63, estrogen receptor, leukocyte common antigen, CD3, CD20, and synaptophysin. Exposure to formic acid had less impact with reduced expression observed for only 3 antibodies (CK8/18, CK7, and TTF-1). The results of this study demonstrate the need to validate immunohistochemical protocols on control tissue treated in the same manner as test tissue, including CytoLyt fixation and exposure of tissue to decalcifying agents.

HPV testing through p16 immunocytochemistry in neck‐mass FNA and its correlation with tissue samples

“…All 8 cases were classified as false‐negative. This discrepancy between cytology and tissue staining may be due to the ethanol‐based fixatives commonly used in cytology smear preparations, which can interfere with immunostaining . All the cytology samples (direct smears) were fixed in ethanol in the current study.…”

Immunohistochemical Analysis of Leucocyte Subsets in the Sinonasal Mucosa of Cats with Upper Respiratory Tract Aspergillosis

“…The sample from the affected cat that was excluded from analysis was collected post mortem and included a large section of nasal turbinates and surrounding osseous structures. It is likely that failure to label this sample was associated with pre-fixation decalcification (Christgau et al, 1998;Gruchy et al, 2015).…”

Detection of Free Cancer Cells in Pelvic Lavage with Double Immunocytochemistry at Rectal Cancer Surgery

“…There was a variable amount of blood in the samples, therefore requiring different amounts of pretreatment with CytoLyt solution, possibly affecting the morphology and the antigenicity of the tumour cells (20,21). We did not make any specific attempt to adjust the number of slides based on the cell density, resulting in a variable cell density in different patients.…”