Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity

doi:10.1016/j.yexmp.2005.10.002

Experimental and Molecular Pathology

Volume 80, Issue 2, April 2006, Pages 183-191

https://doi.org/10.1016/j.yexmp.2005.10.002 Get rights and content

Abstract

Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson’s II, 70% ethanol, UMFIX, modified Carnoy’s, modified methacarn, Bouin’s, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy’s solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy’s solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.

Section snippets

Fixatives

Nine fixatives were used in this study (Table 1). Phosphate-buffered saline (PBS) and 30% sucrose are more accurately termed holding solutions, but for the sake of brevity, they will be grouped with the fixatives in this study. PBS and sucrose were chosen because these are often used to transiently prevent desiccation of tissues that will be subjected to LCM (Parlato et al., 2002).

Tissue preparation

Livers from four adult Sprague–Dawley rats were collected as per Institutional Animal Care and Use Committee

Morphology

Examples of typical morphology for each of the fixatives are shown in Fig. 1. Grading was on a four-point scale, with 1 being poor, and 4 being excellent (Table 3). Tissue was adequate for histologic examinations with all fixatives except Bouin’s, PBS, and 30% sucrose, while modified methacarn had the best morphology. In comparison to standard methods, microwave fixation/microwave processing protocols resulted in modest improvement in morphology for all fixatives without any significant impact

Discussion

The objective of this study was to identify a fixative and an appropriate fixation and processing method that collectively provide the best balance of preservation of tissue morphology and RNA quality/quantity. Using the rat liver as a model, we determined that the fixative is the most critical consideration in optimizing both tissue morphology and RNA integrity.

The morphology associated with each fixative and fixation/processing combination was evaluated based on a 1–4 grading system where

References (24)

H. Abrahamsen et al.
Towards quantitative mRNA analysis in paraffin-embedded tissues using real-time reverse transcriptase-polymerase chain reaction
J. Mol. Diagn.
(2003)
M. Cronin et al.
Measurement of gene expression in archival paraffin-embedded tissues
Am. J. Pathol.
(2004)
J.W. Gillespie et al.
Evaluation of non-formalin tissue fixation for molecular profiling studies
Am. J. Pathol.
(2002)
A. Gloghini et al.
RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues
J. Mol. Diagn.
(2004)
Z.A.M. Hafajee et al.
Ultra-rapid microwave-stimulated tissue processing with a modified protocol incorporating microwave fixation
Pathology
(2004)
M. Koopmans et al.
Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives
J. Virol. Methods
(1993)
R. Parlato et al.
A preservation method that allows recovery of intact RNA from tissues dissected by laser capture microdissection
Anal. Biochem.
(2002)
M.A. Perlmutter et al.
Comparison of snap freezing versus ethanol fixation for gene expression profiling of tissue specimens
J. Mol. Diagn.
(2004)
M. Shibutani et al.
Methacarn: a fixation tool for multipurpose genetic analysis from paraffin-embedded tissues
Methods Enzymol.
(2002)
M. Shibutani et al.
Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens
Lab. Invest.
(2000)

K. Specht et al.

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue

Am. J. Pathol.

(2001)

M. Srinivasan et al.

Effect of fixatives and tissue processing on the content and integrity of nucleic acids

Am. J. Pathol.

(2002)

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Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity

Abstract

Section snippets

Fixatives

Tissue preparation

Morphology

Discussion

J. Mol. Diagn.

Am. J. Pathol.

Am. J. Pathol.

J. Mol. Diagn.

Pathology

J. Virol. Methods

Anal. Biochem.

J. Mol. Diagn.

Methods Enzymol.

Lab. Invest.

Am. J. Pathol.

Am. J. Pathol.