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

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.

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality

“…Moreover, the specific problem of FFPE material needs clarification 31 , 62 - 64 . New RNA quality metrics, which are more sensitive than the RIN values generally used up to now, are recommended to define the preanalytical RNA conditions for reliable expression analyses in future studies 63 , 64 . These are, for example, the DV 200 that represents the percentage of RNA fragments longer than 200 nucleotides or the Q-score that characterizes the ratio of the GAPDH amplicons of 165 bp to 80 bp.…”

Preanalytics and Precision Pathology: Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine

“…Depending on the biomolecule class and the biospecimen type, cold ischemia times of several hours (eg, up to 4-5 hours or even longer at room temperature) have minimal or no detrimental impact on biomolecular quality, extractable quantity, or molecular test results, 14,81,83,[150][151][152] whereas other molecular analyses, sometimes for the same specimen, are altered within 60 minutes or less. ‡ Molecules may be either upregulated or downregulated, and most studies show that gene transcripts are more likely to be upregulated rather than downregulated within 2 hours of cold ischemia.…”

“…80,[158][159][160][161] Current data suggest that the measurable effects of cold ischemia are, at least partially, cancer type-specific as well as biomolecule type-specific and analysis platform-dependent. 80,151 In addition, studies comparing the effects of cold ischemia on cancer tissue and normal tissue from the same organ have demonstrated that tumor tissue is more vulnerable to the effects of cold ischemia. 80,161 Since no single recommendation for cold ischemia time would be optimal for all tissues, all classes of molecules, or all analysis platforms, the recommendation put forward here represents a compromise that meets most current molecular testing needs for cancer patient specimens.…”

Effects of ex�vivo ischemia time and delayed processing on quality of specimens in tissue biobank

“…There are various factors influencing the quality of biospecimens, and pre-analytical variables are crucial ( 5 , 6 ). Pre-analytical variables are divided into the pre-acquisition and post-acquisition phases.…”