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Arch Pathol Lab Med. 2019 Sep;143(9):1106-1118. doi: 10.5858/arpa.2018-0172-OA. Epub 2019 Feb 20.

The Biospecimen Preanalytical Variables Program: A Multiassay Comparison of Effects of Delay to Fixation and Fixation Duration on Nucleic Acid Quality.

Author information

1
From Biorepositories and Biospecimen Research Branch, Cancer Diagnosis Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland (Drs Carithers, Guan, Odeh, Sachs, Branton, and Moore); Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland (Drs Agarwal, Barcus, Lih, Williams, Sobin, Roche, and Mr Soria); Preferred Solutions Group, Preferred Scientific Group, Washington, District of Columbia (Dr Engel); Kelly Government Solutions, Kelly Services, Rockville, Maryland (Dr Greytak and Mr Fombonne); the Department of Pathology, University of New Mexico, Albuquerque (Dr Bocklage); the Department of Pathology and Laboratory Medicine, Boston Medical Center, Boston, Massachusetts (Dr Andry and Ms Duffy); the Department of Pathology and Laboratory Medicine, Emory University, Winship Cancer Institute, Atlanta, Georgia (Dr Sica); the Department of Pathology, University of Pittsburg, Pittsburgh, Pennsylvania (Dr Dhir); and Van Andel Research Institute, Pathology and Biorepository Core, VARI Core Technologies and Services, Grand Rapids, Michigan (Dr Jewell).

Abstract

CONTEXT.—:

Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown.

OBJECTIVE.—:

To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program.

DESIGN.—:

The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls.

RESULTS.—:

DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point-specific effects of DTF and TIF. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV200 (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay.

CONCLUSIONS.—:

DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

PMID:
30785788
DOI:
10.5858/arpa.2018-0172-OA

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