The Biospecimen Preanalytical Variables Program: A Multiassay Comparison of Effects of Delay to Fixation and Fixation Duration on Nucleic Acid Quality.
Author(s): Carithers LJ, Agarwal R, Guan P, Odeh H, Sachs MC, Engel KB, Greytak SR, Barcus M, Soria C, Lih CJ, Williams PM, Branton PA, Sobin L, Fombonne B, Bocklage T, Andry C, Duffy ER, Sica G, Dhir R, Jewell S, Roche N, Moore HM
Publication: Arch Pathol Lab Med, 2019, Vol. , Page
PubMed ID: 30785788 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to assess effects introduced by a delay to fixation (cold ischemia time) and the duration of formalin fixation on the quantity and quality (integrity) of DNA and RNA isolated from prospectively collected formalin-fixed, paraffin-embedded (FFPE) colon, kidney, and ovarian tumor specimens under the National Cancer Institute's Pre-analytical Variable (BPV) Program. Results obtained with FFPE specimens were compared to case-matched snap-frozen controls. RNA quality assessment methods were also compared.
Conclusion of Paper
The authors advise that each FFPE block be screened for tumor and necrosis content, as a representative quality control (QC) FFPE block was insufficient at predicting histomorphology in approximately 40% of cases analyzed. DNA and RNA quantity were not influenced by a delay to fixation (DTF) of up to 12 h at room temperature or a time in fixative (TIF) of up to 72 h. Conversely, a DTF of 12 h and a TIF of 72 h elicited significant differences in DNA integrity as evidenced by changes in a qPCR-based DNA quality assay and in some RNA quality assays when compared to shorter durations. When RNA quality assays were compared, the qRT-PCR-based Q-score consistently revealed differences between FFPE and snap-frozen specimens, and among respective DTF and TIF timepoints. Conversely, RNA integrity number (RIN) revealed differences between FFPE and snap-frozen specimens but not DTF and TIF timepoints, and differences in DV200 values were largely dependent on experimental factor (preservation method, DTF, TIF), tumor type, and experimental timepoint.
Studies
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Study Purpose
The purpose of this study was to determine the extent that DNA and RNA quantity and quality (integrity) are affected by a delay to fixation and the duration of formalin fixation in FFPE tumor specimens. The BPV Program's Scientific Steering Committee identified delay to fixation (DTF) and time in fixative (TIF) as pre-analytical factors requiring additional study. To address this, primary tumor specimens were collected from patients diagnosed with renal cell carcinoma (kidney), colorectal adenocarcinoma (colon), and ovarian/fallopian tube carcinoma (ovary) at four different medical centers. The effects of DTF were investigated in kidney, ovary, and colon tumor aliquots subjected to a room temperature delay of 1, 2, 3, or 12 h in a humidified chamber before fixation in 10% neutral buffered formalin (NBF) for 10-12 h. Effects of TIF were investigated in kidney and ovary tumor aliquots fixed in 10% NBF at room temperature for 6, 12, 23 or 72 h within 1 h of collection. Two additional tumor aliquots were collected from each patient and preserved within 1 hour of collection and either snap-frozen in liquid nitrogen (LN2) and stored in LN2 vapor for use as a gold standard, or fixed in 10% NBF at room temperature for 23 h to verify that cases met histopathology eligibility criteria (>50% tumor content by surface area, <20% necrosis); although eligibility was ultimately determined for each FFPE block. RNA and DNA were extracted from FFPE sections using the QIASymphony RNA kit and QIASymphony DNA mini kit, respectively, albeit different protocols were used for snap-frozen and FFPE specimens. Nucleic acid concentration, purity, and potential contamination were assessed by Nanodrop spectrophotometry. DNA and RNA quantity was determined by fluorometry with corresponding Qubit kits. DNA quality (integrity) was determined by the qPCR-based KAPA Human Genomic DNA Quantification and QC kit and reported as a Q-ratio, a ratio of medium (129 bp) or long (305 bp) length amplicons relative to a short (41 bp) amplicon for a conserved single-copy gene. RNA quality (integrity) was assessed by RIN and DV200 scores that were generated with an Agilent bioanalyzer. RNA quality (integrity) was also assessed by a qRT-PCR-based assay and reported as a Q-score, a ratio of medium (165 bp) to short (80 bp) amplicons of GAPDH or PGK1. Although the number of specimens analyzed depended on the experimental timepoint and assay, 17 to 29 kidney, 4-10 ovary, and 9-13 colon tumor specimens were analyzed. All experimental timepoints were randomly assigned and researchers were blind to these assignments during analysis.
Summary of Findings:
Although more than 4,000 patients were screened, when pre- and post-surgical eligibility criteria were applied tumor specimens were collected and processed from 203 and 113 patients for DTF and TIF experiments, respectively.When the representative QC FFPE block was assessed, 84% of the 300 patient cases passed histological criteria (≥50% tumor content by surface area, <20% necrosis); however, only 59% of these 252 cases passed when all four experimental blocks were assessed. This led the authors to conclude that evaluation of a single representative FFPE block is insufficient, and tumor and necrosis contents should be determined for each FFPE block.
The quantity of DNA and RNA isolated from FFPE specimens was not influenced by a DTF of up to 12 h at room temperature or a TIF of up to 72 h in any of the tumor types examined. When DNA quality was assessed by a qPCR-based assay, FFPE blocks displayed significantly lower mean Q-ratios than case-matched snap-frozen controls (p<0.001) regardless of DTF, TIF, or tumor type, with the exception of TIF ovary specimens (p=0.06). Among FFPE experimental blocks, mean Q-ratios were significantly higher among 12 h DTF kidney, ovary, and colon tumor specimen and significantly lower among 72 h TIF kidney specimens compared to shorter timepoints; however, differences between TIF timepoints were nonsignificant in ovary specimens.
RNA quality assays differed in their ability to detect differences among FFPE tumor specimens and snap-frozen controls, and among FFPE DTF and TIF timepoints. FFPE tumor specimens displayed consistent and significantly lower RINs (>7 versus <3; p<0.001) and qRT-PCR-based Q-scores (p<0.001) than snap-frozen case-matched control specimens. However, significant differences in DV200 values between FFPE and snap-frozen specimens were limited to DTF ovary specimens (p=0.009), TIF kidney (p<0.001), and TIF ovary specimens (p=0.04), with nonsignificant differences observed among DTF ovary and colon specimens. Significant differences in RNA quality (integrity) among DTF and TIF timepoints were dependent upon the assay, tumor type and experimental timepoint. RIN was not affected by DTF or TIF with the exception of DTF colon specimens (p=0.03). DV200 values were significantly lower among 12h DTF kidney specimens (p=0.02) and 72 h TIF kidney specimens (p<0.001) compared to the collective mean of the other timepoints. Conversely, significant differences in qRT-PCR-based Q-scores were consistently detected across tissue types for DTF timepoints (p=0.03 for colon; p<0.001 for kidney and ovary), as well as for TIF kidney specimens (p<0.001). When significant differences were observed, Q-scores were higher in 12 h DTF specimens but lower in 72 h TIF specimens, compared to the collective mean of shorter timepoints.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Fluorometry DNA Real-time qPCR Morphology H-and-E microscopy RNA Fluorometry RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 1 h
2 h
3 h
12 h
Real-time qRT-PCR Specific Targeted nucleic acid GAPDH
PGK1
Biospecimen Preservation Time in fixative 6 h
12 h
23 h
72 h