Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Optimization of Pre-Analytical Handling to Maintain DNA Integrity in Diagnostic Papanicolaou Tests.

Author(s): Schumacher S, Malchau Lauesgaard J, Carlsson T, Linder A, Sundfeldt K

Publication: J Mol Diagn, 2025, Vol. 27, Page 199-208

PubMed ID: 39828035 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how the storage of cervical cytology specimens in ThinPrep solution, which were collected for Papanicolaou (Pap) testing, can affect DNA integrity and size profiles. The percentage of DNA fragments that were 100-2500 bp and 100-350 bp in size were compared among ovarian cell culture specimens that were stored for 48 h at room temperature followed by either 0 h (control), >4 weeks at 4°C, or 2 months at -20°C or -80°C; among ovarian cell culture specimens stored for up to 48 h at room temperature; and among prospectively collected cervical cytology specimens that were stored for <4 h or 48 h at room temperature or 4°C.  Twelve archival cervical specimens were collected using a cytobrush from patients with ovarian cancer and stored in ThinPrep PreservCyt solution for 48 h at room temperature before storage at -80°C for 64-79 months. Duplicate cervical  specimens were collected using a cytobrush from 10 patients with an unspecified benign gynecologic condition, stored in ThinPrep PreservCyt solution for <4 h at room temperature and 4°C, centrifuged at 200 g for 10 minutes, and DNA was isolated from the half of the cell pellet immediately and the other half was resuspended in supernatant and stored for a total of 48 h at room temperature and 4°C.  Cultured cells from a human ovarian carcinoma cell line (SK-OV-3) were placed in ThinPrep PreservCyt and either stored at room temperature for 48 h followed by storage for 0 h (control), 4 weeks at 4°C, or ≥2 months at -20°C or -80°C before DNA extraction or stored at room temperature for 0, 6, 12, 24, 36, and 48 h or 4°C for 0, 6, 12, 24, 36, 48, 60, 72, 84, and 96 h before extraction. Collected cervical specimens and cultured cells were pelleted by centrifugation at 200 g for 10 minutes and DNA was extracted from the pellet using the QIAmp DNA Micro Kit and the supernatant using the QIAmp Circulating Nucleic Acid Kit. DNA was quantified using the Qubit dsDNA High Sensitivity Assay Kit. DNA fragment size analysis was conducted using the High Sensitivity D5000 ScreenTape Assay and Genomic DNA ScreenTape Assay on a 4200 TapeStation instrument.

   Summary of Findings:

   DNA yield was higher from cell pellets than supernatants of both archival (586.05 ng versus 33.08 ng) and prospectively collected PapTests (1.61 versus 0.06 µg). Short (<2500 bp) DNA fragments with a peak at <300 bp were detected in all twelve pellets from archival cervical cytology specimens stored at room temperature for ≥48 h prior to storage at -80°C for 5-6 years, and in five of the supernatants.  Short DNA fragments with a mean size of 269 bp were observed in all specimens from a cultured ovarian cell line preserved in ThinPrep solution, but the specimens stored at 4°C for 4 weeks after initial storage for 48 h at room temperature had significantly more fragments that were 100 -2500 bp (P=0.013) and 100-350 bp (P= 0.026) in size and displayed a trend toward a lower DIN than control specimens that were just stored for 48 h at room temperature.  Although specimens from a cultured ovarian cell line that were stored at -20°C or -80°C for ≥2 months after the initial storage for 48 h at room temperature had more fragments that were 100 -2500 bp and 100-350 bp in size and a lower DIN than control specimens stored for 48 h at room temperature; the differences were not significant.  The percentage of 100-2500 bp and 100-350 bp DNA fragments increased when cultured cells were preserved and stored in ThinPrep solution; significant increases relative to the control (0 h) were observed after storage at 36 h at room temperature (P<0.05), after 48 h at either room temperature (4.2-fold, P=0.003 and 11.5-fold, P=0.002, respectively) or 4°C (1.5-fold, P=0.002 and 1.7-fold, P=0.03, respectively) and after 96 h at 4°C (room temperature not investigated, 1.9-fold and 3.6-fold, respectively, P<0.05, both). In cultured cells from an ovarian cell line, the increase in the percentage of 100-2500 bp and 100-350 bp DNA fragments was significantly higher when specimens were stored in ThinPrep solution for 48 h at room temperature than 4°C (P=0.029, both).  The percentage of fragments that were 100-230 bp, 230-350 bp and 100-2500 bp were higher in DNA isolated from the cell pellet of prospectively collected ThinPrep-preserved cytology specimens stored for 48 h at room temperature or 4°C than case-matched specimens stored for < 4 h. However, those stored in ThinPrep at room temperature for 48 h had a larger increase in the percentage of DNA fragments between 100 to 2500 bp (P=0.03), 100 to 230 bp (P=0.02), and 230 to 350 bp (P=0.007) than those stored at 4°C for 48 h. While DIN decreased when prospectively collected ThinPrep-preserved cytology specimens were stored at room temperature or 4°C for 48 h versus < 4 h (6.2 versus 7.4 and 6.6 versus 7.1, respectively), the trend toward larger decrease in DIN with room temperature storage was not significant.

   Biospecimens

   • Cell - Ovary

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Capillary electrophoresis-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	Cultured cells at room temperature for 0, 6, 12, 24, 36, and 48 h Cultured cells at 4°C for 0, 6 , 12, 24, 36, 48, 60, 72, 84, and 96 Cultured cells for 48 h at room temperature followed by 0 h versus 4 weeks at 4°C or ≥ 2 months at -20°C or -80°C PapTests for <4 h versus 48 h at 4°C and room temperature
   Storage	Storage temperature	4°C -20°C -80°C Room temperature vs 4°C

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