Number

093-EN

Section

General Section

Use

Abstract

70% ethanol fixation and paraffin embedding is a useful alternative method for molecular profiling studies. This was shown during a systematic approach of processing clinical tissue specimen (prostate and kidney) through non-formalin fixation method.

Substituted substances

Alternative Substances

Reason substitution

CMR
skin/respiratory sensitizing

Other type of alternative

Snap freezing

Hazard Assessment

Substance to be substituted: Formaldehyde is in the database of hazardous substances according to SUBSPORTplus criteria. Formaldehyde is a sensitiser, may cause cancer, is toxic if swallowed, is toxic in contact with skin, causes severe skin burns and eye damage, is toxic if inhaled and is suspected of causing genetic defects. Alternative substance: Ethanol is flammable and a group 1 carcinogen (IARC), as listed in the Substance Database according to SUBSPORTplus Screening Criteria (SDSC). The hazard of the non-chemical alternative is not assessed in SUBSPORTplus.

Description of Substitution

Concerning histology the aim of the study was to use a systematic approach to evaluate the biomolecular status of a number of clinical tissue specimens processed through a non-formalin fixation method. 50 radical prostatectomies (tissue from prostate) were fixed in 70% ethanol and studied at the National Cancer Institute. In five of these cases, the specimen was subdivided and processed through four separate methods (snap freezing, ethanol fixation, paraffin embedding) and these four methods were compared. The conclusion was, that clinical diagnosis was possible with all methods. Also ethanol fixation was comparable to formalin fixation and superior to snap freezing. The polyester wax was difficult to use for large specimens. Following you will find a citation of the summary from the article: "Many commercially available antibodies have been selected for use on formalin fixed tissue. The investigators therefore have to assess the performance using ethanol instead. Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analysed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. "

Case/substitution evaluation

This study shows that there are technical feasible and safer alternatives to formaldehyde. The substitute ethanol is suitable in this case although it is carcinogenic, it is still less harmful to human health.

Availability of Alternative

Available

Type of information supplier

Research

Type of publication and availability

Freely available from link

Publication source: author, company, institute, year

Gillespie John W. et al: Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies. American Journal of Pathology. Vol. 160. No 2. February 2002. 449-457

Publication source

Type of publication and availability

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1850633/

Date, reviewed

December 11, 2020