Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Cold Formalin Fixation Guarantees DNA Integrity in Formalin Fixed Paraffin Embedded Tissues: Premises for a Better Quality of Diagnostic and Experimental Pathology With a Specific Impact on Breast Cancer.

Author(s): Berrino E, Annaratone L, Miglio U, Maldi E, Piccinelli C, Peano E, Balmativola D, Cassoni P, Pisacane A, Sarotto I, Venesio T, Sapino A, Marchiò C

Publication: Front Oncol, 2020, Vol. 10, Page 173

PubMed ID: 32140450 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare DNA yield, purity and integrity in case-matched cold- and room temperature fixed FFPE tumor specimens.  The study included both prospectively collected specimens and those stored for 6 years. To further investigate the effects of storage, a second extraction was performed from some prospectively collected specimens 6 months after the initial extraction. Specimens were obtained from thirty-eight surgical specimens under-vacuum packing and cooling. Matched specimens were divided and one tissue segment was fixed in 4% neutral buffered formalin (NBF) for 24 h at room temperature and the other was fixed in 4% NBF at 4°C for 24 h. The specimen fixed at 4°C was dehydrated in 95% ethanol at 4°C, after which the remainder of dehydration, clearing, and paraffin embedding was performed using the same automated processor at room temperature for the two specimen sets. The first 21 pairs were collected 6 years prior to analysis and included 11 breast carcinomas, three colorectal adenocarcinomas, three lung adenocarcinoma, two gastric adenocarcinomas, and two thyroid follicular carcinomas. The remaining 17 pairs were collected prospectively and included eight colorectal adenocarcinoma, two gastro-intestinal stromal tumors, two lung adenocarcinomas, one intrahepatic cholangiocarcinoma, one splenic metastasis of endometrial carcinoma, one serous-papillary ovarian carcinoma, one pleomorphic undifferentiated sarcoma and one gallbladder adenocarcinoma. Tumor area identified from a representative hematoxylin and eosin stained slide was macrodissected from five 8 µm sections and used for DNA extraction with the QIAamp DNA FFPE Tissue Kit. A second extraction was performed after 6 months from 14 of the prospectively collected specimens.  DNA was quantified using the high sensitivity DNA kit on a Qubit 3.0 Fluorimeter and by spectrophotometer. DNA fragment size was evaluated using the real-time PCR based DEPArray FFPE QC Kit.

   Summary of Findings:

   DNA yields were higher from  specimens fixed at 4°C (cold-fixed) than those fixed at room temperature, regardless of analysis method (4.9 µg versus 4.2 µg by fluorometry, 20.37 µg versus 16.2 µg by spectrophotometer), but the difference was only significant among archival specimens analyzed by fluorometry (4.1 µg versus 2.1 µg, P=0.04). Compared to prospectively collected specimens, specimens archived for 6 y yielded 40% less DNA when cold-fixed (P=0.03) and 70% less DNA when fixed at room temperature (P<0.001). When prospectively collected and archival specimens were analyzed together, the mean QC score in the real-time PCR-based fragmentation assay was significantly lower (indicating more fragmentation) for room temperature-fixed than cold-fixed specimens (0.36 versus 0.69, P<0.0001) with a 6-fold difference observed in archival specimens (P<0.0001), and a much smaller difference found in prospectively collected specimens (P=0.0016). Further examination showed that cold-fixed specimens generated comparable QC scores between prospectively collected and archival specimens, while specimens fixed at room temperature generated a significantly lower mean QC score in archival FFPE blocks than prospectively collected specimens (P<0.0001). Further indicating a greater susceptibility of room temperature fixed FFPE blocks to storage, QC scores were significantly lower in DNA obtained in a second extraction from prospectively collected room temperature-fixed specimens than in the initial extraction (~35% lower, P<0.0001), but QC scores were comparable between extractions for prospectively collected specimens fixed at 4°C (P=0.131). The difference in QC scores between room temperature and cold-fixation was larger in breast carcinoma specimens than colon or lung specimens, indicating a tissue-specific sensitivity. The authors state the pathologists did not report any fixation artifacts. There was no effect of fixation temperature on the purity (OD 230/260 and OD 260/280) of the DNA obtained.

   Biospecimens

   • Tissue - Breast
   • Tissue - Colorectal
   • Tissue - Lung
   • Tissue - Gall Bladder
   • Tissue - Ovary
   • Tissue - Uterus
   • Tissue - Stomach

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Sarcoma
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Fluorometry
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Breast Lung Colon
   Storage	Storage duration	0 years 6 months 6 years
   Biospecimen Preservation	Temperature of fixation/preservation	4°C Room temperature

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