Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Critical steps in tissue processing in histopathology.

Author(s): Comanescu M, Annaratone L, D'Armento G, Cardos G, Sapino A, Bussolati G

Publication: Recent Pat DNA Gene Seq, 2012, Vol. 6, Page 22-32

PubMed ID: 22208680 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of cold ischemia time on RNA integrity number (RIN) values in unfixed specimens and to determine the effects of formalin fixation temperature on mRNA expression by whole genome array analysis and real time qRT-PCR with a comparison to frozen specimens.

Conclusion of Paper

RIN values remained acceptable (all >7), regardless of cold ischemia time (1-72 h) when unfixed specimens were sealed in plastic bags under-vacuum and cooled to 4 degrees C immediately after procurement. Cycle threshold (Ct) values were slightly higher for formalin-fixed paraffin-embedded (FFPE) tissue fixed at room temperature than for FFPE tissue fixed at 4 degrees C, but frozen tissue gave the lowest Ct values. No significant differences were noted in the normalized gene expression levels between differently preserved specimens. Whole genome array analysis revealed that a similar number of transcripts were detected in RNA extracted from FFPE tissue fixed at 4 degrees C and from frozen tissue, but the lists were not entirely overlapping. Formalin fixation at 4 degrees C allowed for the amplification of RNA fragments up to 660 bp long, which the authors report was an improvement over room temperature-fixed FFPE specimens. The authors also report that microarray gene expression analysis was similar between cold fixed FFPE and frozen specimens.

Studies

Study Purpose

The purpose of this study was to determine the effects of cold ischemia time at 4 degrees C for specimens that were sealed in plastic bags under-vacuum and cooled immediately after procurement on RIN values.

Summary of Findings:

RIN values remained acceptable (all >7), regardless of cold ischemia time for specimens that were sealed in plastic bags under-vacuum and cooled to 4 degrees C immediately after procurement. The average RIN values after cold ischemia times of 1-4 h, 5 h, 6-23 h, 24-48 h, and 60-72 h were 8.2, 7.8, 8.0, 7.8, and 7.5, respectively.

Biospecimens

Tissue - Breast

Preservative Types

None (Fresh)

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
RNA	Automated electrophoresis/Bioanalyzer

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Acquisition	Cold ischemia time	1-4 h 5 h 6-23 h 24-48 h 60-72 h

Study Purpose

The purpose of this study was to determine the effects of formalin fixation temperature on mRNA levels of 84 genes using real time qRT-PCR with the Human Breast Cancer RT2 Profiler PCR Array and whole genome array analysis. Frozen tissue was also included for comparison.

Summary of Findings:

Ct values were slightly higher for FFPE tissue fixed at room temperature than for FFPE tissue fixed at 4 degrees C, but frozen tissue gave the smallest Ct values. However, no significant differences were noted in the normalized expression levels of the 84 genes included in the RT-PCR array between differently preserved specimens. A similar number of transcripts were detected on a whole genome array using RNA extracted from FFPE tissue fixed at 4 degrees C and from frozen tissue (a comparison with room temperature fixed FFPE tissue was not reported). 25,852 transcripts were detected in both types of specimens while 3,874 and 2,649 transcripts were only detected in the frozen and cold-fixed FFPE specimens, respectively.

Biospecimens

Tissue - Breast

Preservative Types

Formalin
Frozen

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
RNA	Real-time qRT-PCR
RNA	DNA microarray
RNA	Low density array

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
Biospecimen Preservation	Temperature of fixation/preservation	Room temperature 4 degrees C

Study Purpose

The purpose of this study was to determine the effects of formalin fixation temperature on RT-PCR amplification of different sized fragments from colon, breast, pancreas, and gastric cancer specimens. Results were compared with frozen specimens. Gene expression profiling was also performed using microarray analysis.

Summary of Findings:

Formalin fixation at 4 degrees C allowed for the amplification of RNA fragments up to 660 bp long, which the authors report was an improvement over room temperature-fixed FFPE specimens. The authors also report that RNA from cold fixed FFPE specimens was significantly less fragmented and contained higher amounts of more than 500 base long fragments than RNA from room temperature fixed FFPE specimens. Further, the authors state that microarray gene expression analysis was similar between cold fixed FFPE and frozen specimens.

Biospecimens

Preservative Types

Formalin
Frozen

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
RNA	RT-PCR
RNA	DNA microarray

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
Biospecimen Preservation	Temperature of fixation/preservation	Room temperature 4 degrees C
RT-PCR Specific	Targeted nucleic acid	PBGD ABL B2M G6PD CK20 mammaglobin ER alpha
RT-PCR Specific	Length of gene fragment	128 bp 193 bp 329 bp 331 bp 334 bp 346 bp 500 bp 660 bp

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