Evaluating Pre-Analytical Variables for Saliva Cell-Free DNA Liquid Biopsy.
Author(s): Salfer B, Havo D, Kuppinger S, Wong DTW, Li F, Zhang L
Publication: Diagnostics (Basel), 2023, Vol. 13, Page
PubMed ID: 37238150 PubMed Review Paper? No
Purpose of Paper
This paper compared the yield of cell-free DNA (cfDNA) in saliva preserved with EDTA (0, 5, 10 and 20 mM), Streck Urine Preserve, and Nonosanis UAS and stored at room temperature before centrifugation for up to 7 days. The effect of collection into the Oasis Pure Sal collectors instead of Falcon tubes and storing centrifuged saliva with 0, 2 and 20 mM EDTA at room temperature for up to 7 days was also investigated.
Conclusion of Paper
Saliva cfDNA yields declined rapidly when specimens were stored at room temperature, but the decline was attenuated by the addition of 5-20 mM EDTA upon collection or after centrifugation. When preservation methods were compared, saliva treated with Streck Urine Preservative resulted in recovery of excess cfDNA and yields further increased with storage. When saliva was preserved with Nonosanis UAS preservative, DNA recovery was less than half of that recovered from unpreserved saliva. The half-life of saliva cfDNA in the absence of EDTA was 13.18 h when stored after centrifugation and 13.94 h when stored prior to centrifugation. cfDNA yield decreased when centrifuged saliva was stored in the absence of EDTA or when 2 mM EDTA was added but was stabilized by the addition of 20 mM EDTA. Significantly less cfDNA was recovered from saliva specimens collected in Oasis Pure Sal collectors than when Falcon tubes were used for collection.
Studies
-
Study Purpose
This study compared the yield of cell-free DNA (cfDNA) in saliva preserved with EDTA (0, 5, 10 and 20 mM), Streck Urine Preservative, and Nonosanis UAS preservative and stored at room temperature for up to 7 days before centrifugation, extraction, and analysis. Potential effects of saliva collection in Oasis Pure Sal collectors instead of Falcon tubes and storing centrifuged saliva with 0, 2 or 20 mM EDTA at room temperature for up to 7 days were also investigated. Unless otherwise specified, saliva was collected from an unspecified number of healthy volunteers into chilled falcon tubes, pooled, vortexed for 10-15 s and aliquoted. Unless otherwise specified, saliva was immediately centrifuged at 2600 g for 15 min at 4°C. cfDNA was extracted using the Qiagen Circulating Nucleic Acid Kit and stored at -20°C. EGFR was quantified by droplet digital PCR. To investigate potential effects associated with collection container, matched saliva from healthy volunteers was collected in Oasis Pure SAL collectors and Falcon tubes without EDTA. To investigate potential effects of preserving saliva with EDTA, aliquots of pooled saliva from healthy volunteers were collected in Falcon tubes containing 0, 2 or 20 mM EDTA, centrifuged immediately and stored at room temperature for 0, 1, 4, and 7 days before analysis. To study the stability of cfDNA in uncentrifuged saliva, saliva aliquots containing 5, 10, and 20 mM EDTA were stored at room temperature for 1, 4 or 7 days before centrifugation and DNA extraction. To test the effect of preservation with Streck Urine Preservative, the preservative was added to saliva at a ratio of 1:10 and cfDNA was extracted after 0, 1, 4 and 7 days. To test the effect of preservation with Nonosanis UAS preservative, UAS preservative was added to saliva at a ratio of 2:5 and cfDNA was extracted after 0, 1, 4 and 7 days.
Summary of Findings:
Saliva collected in Oasis Pure Sal collectors instead of Falcon tubes resulted in recovery of only 8% of cfDNA (P<0.001). In urine collected in Falcon tubes, cfDNA yield decreased similarly over time in the absence of EDTA (to 43% and 18% of baseline by day 1 and 7, respectively) and when 2 mM EDTA was added (to 52% and 22% of baseline by day 1 and 7, respectively), but increased insignificantly when 20 mM EDTA was added (to 121.9% and 126.6% of baseline by day 1 and 7, respectively). The half-life of centrifuged saliva cfDNA in the absence of EDTA was calculated to be 13.18 h. When saliva was stored at room temperature prior to centrifugation without EDTA, the cfDNA yield decreased to 32% and 2% of baseline on day 1 and 7, respectively. The half-life of cfDNA in uncentrifuged saliva was calculated to be 13.94 h. When uncentrifuged saliva was preserved with 5, 10 or 20 mM EDTA, the cfDNA yield was 8-, 14- and 6-fold higher, respectively on day 4 than when no EDTA was added. When Streck Urine Preservative was added to saliva from Falcon tubes, cfDNA yield was twice that of unpreserved saliva and doubled over the course of 7 days of room temperature storage. In contrast, addition of Nonosanis UAS preservative to saliva resulted in recovery of only 43% of the DNA recovered from unpreserved saliva, although DNA levels declined only non-significantly with further storage.
Biospecimens
Preservative Types
- None (Fresh)
- Other Preservative
- Streck/BCT
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Falcon tube
Oasis Pure Sal collectors
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage conditions Centrifuged
Uncentrifuged
Biospecimen Preservation Concentration of fixative 0 mM
2 mM
5 mM
10 mM
20 mM
Storage Storage duration 0 days
1 day
4 days
7 days
Biospecimen Preservation Type of fixation/preservation EDTA
Streck cfDNA urine presevative
UAS
None (fresh)
Refrigeration