Evaluation of the Value of Frozen Tissue Section Used as “Gold Standard” for Immunohistochemistry

Abstract

To examine the use of acetone- or ethanol-fixed frozen tissue sections as the "gold standard" for immunohistochemical analysis, we evaluated frozen sections with various conditions of fixation and antigen retrieval (AR). Fresh human tissues were frozen in OCT. An adjacent tissue block was fixed in 10% neutral buffered formalin (NBF) and paraffin embedded (FFPE). Frozen sections were fixed by 6 protocols: acetone, ethanol, NBF (2 durations), and NBF + calcium chloride (2 durations). AR was used for all NBF-fixed sections. More than half of the antibodies (16/26 [62%]) showed immunohistochemical results indistinguishable between acetone- and NBF-fixed sections; 8 (31%) showed better immunohistochemical signals following NBF and AR; 2 gave better immunohistochemical results for acetone-fixed sections. Most cytoplasmic proteins (10/13) showed comparable immunohistochemical signals between acetone- and NBF-fixed sections. For nuclear proteins, NBF-fixed sections gave better immunohistochemical signals than did acetone-fixed sections. In most cases, NBF yielded stronger signals with less background and better morphology. The data do not support the use of acetone-fixed frozen tissue sections as the gold standard for immunohistochemical analysis. In evaluating new antibodies, a combination of acetone- and NBF-fixed frozen sections should be used, although in practice, FFPE tissue sections may serve as the standard for most antigens for immunohistochemical analysis.

Inconsistent Immunohistochemical Expression Patterns of Four Different CD133 Antibody Clones in Glioblastoma

“…To obtain comparable and reproducible results, a series of exclusion criteria for used human subject tissue was defined. Glioblastomas where histological material had been frozen before paraffin embedding were excluded because the morphology of frozen tissue is compromised compared to the morphology of paraffin-embedded tissue (Shi et al 2008). Because of pronounced tumor heterogeneity, it was not optimal to assess CD133 expression in small biopsy specimens.…”

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

“…While formaldehyde fixation preserves tissue architecture and allows tissue storage and transport at RT, it also inactivates enzymes and crosslinks proteins necessitating further steps to achieve accurate immunostaining. This problem is eliminated with the use of frozen sections but this method presents unique technical challenges in obtaining optimal staining results and accurate interpretation 14,15 .…”

“…The fixation step helps to preserve morphology better but also cross links endogenous proteins thus necessitating intricate antigen retrieval procedures for immunostaining and eliminating the possibility of enzymatic staining on such sections 12 . Frozen sections, on the other hand, do not need to be pre-fixed or processed; therefore, proteins are able to retain native biological conformations 13,14 . Further, frozen sections also allow for quick turnaround time, which at times is necessary, for example in the intraoperative diagnosis of sarcomas and other surgical biopsies 15 .…”

The Effect of Freezing on the Immunoprofile of Breast Carcinoma Cells

“…), it is commonly believed that fixation for 8-36 hours is required to obtain the best immunohistochemical results (15)(16)(17)(18). As is already known, loss of immunoreactivity can be seen after "coagulant" fixatives like acetone and ethanol are used (19,20). The mechanism of tissue fixation is not well understood, but it is stated that coagulating fixatives lead to dehydration, which decomposes the protein structure by removing free water from the tissue.…”