Ultrasound-accelerated formalin fixation improves the preservation of nucleic acids extraction in histological sections.

Author(s): Fracasso T, Heinrich M, Hohoff C, Brinkmann B, Pfeiffer H

Publication: Int J Legal Med, 2009, Vol. 123, Page 521-5

PubMed ID: 19685069 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to investigate the ability of ultrasound-accelerated formalin fixation (UAFF) to increase the quality of preservation of antigens and nucleic acids in autopsy specimens due to shorter tissue exposure time to formaldehyde.

Conclusion of Paper

The use of UAFF resulted in no differences with H&E staining or immunohistochemical staining when compared to specimens fixed using conventional buffered formalin fixation. RNA yield and quality significantly increased with UAFF. Furthermore, the highest yield and best quality RNA was extracted from specimens with the shortest fixation time (15 min versus 30 min). The authors note, however, that RNA yield data was obtained after normalization against amount of tissue and should be considered preliminary data. While DNA yield and quality was not as dramatically affected, the conventional method of fixation did result in more frequent allele dropout, and better amplification of longer amplicons was seen from UAFF-derived samples.

Studies

Study Purpose

The purpose of this study was to see if UAFF for either 15 or 30 min improved DNA quality or quantity extracted from autopsy specimens of heart, liver, lung, kidney, and spleen when compared to that of conventional formalin fixation.

Summary of Findings:

The amount of DNA extracted did not appear to be dependent on the method of fixation. However, the quality of DNA, as determined by PCR amplification using the Mentype NonaplexQS kit from Biotype, was determined to be slightly better from specimens with shorter fixation times (UAFF, 15 min or 30 min), specifically for longer amplicons. Differences between the two UAFF times were absent with the exception of one amplification locus where the shorter fixation time yielded better results.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Autopsy

Platform:

Analyte	Technology Platform
DNA	PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Preservation	Time in fixative	15 min 30 min 24-48 h
Biospecimen Preservation	Method of fixative delivery	Immersion Ultrasound-accelerated

Study Purpose

The purpose of this study was to see if UAFF for either 15 or 30 min improved RNA quality or quantity extracted from autopsy specimens of heart, liver, brain, kidney, and spleen when compared to that of conventional formalin fixation.

Summary of Findings:

RNA quality consistently improved with the shorter fixation time (15 min UAFF). All three GAPDH fragments were able to be amplified from specimens fixed for 15 min, however, with a fixation time of 30 min or with conventional formalin fixation, only the shorter fragments could be amplified. Notably, there were only slight differences observed between specimens fixed for 30 min or 24-48 h. While RNA yield also improved with shorter fixation time, data was normalized against the amount of tissue and therefore should be considered preliminary results.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Autopsy

Platform:

Analyte	Technology Platform
RNA	Real-time qRT-PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Preservation	Time in fixative	15 min 30 min 24-48 h
Biospecimen Preservation	Method of fixative delivery	Immersion Ultrasound-accelerated
Real-time qRT-PCR Specific	Targeted nucleic acid	GAPDH
Real-time qRT-PCR Specific	Length of gene fragment	114 bp 233 bp 453 bp

Study Purpose

The purpose of this study was to see if UAFF might affect the quality of H & E staining of autopsy specimens from heart, lung, brain, pancreas, kidney, spleen, and liver, or histochemical staining of lung specimens, when compared to conventional formalin fixation.

Summary of Findings:

The shorter fixation times used during UAFF did not result in loss of quality of H & E staining or immunohistochemical staining.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Autopsy

Platform:

Analyte	Technology Platform
Morphology	H-and-E microscopy
Protein	Immunohistochemistry

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Preservation	Time in fixative	15 min 30 min 24-48 h
Biospecimen Preservation	Method of fixative delivery	Immersion Ultrasound-accelerated
Immunohistochemistry Specific	Targeted peptide/protein	Actin CD68

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