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Prospective Cohort Study to Assess the Effect of Storage Duration, Leuko-Filtration, and Gamma Irradiation on Cell-Free DNA in Red Cell Components.

Author(s): Gupta N, Khetan D, Chaudhary R, Shukla JS

Publication: Transfus Med Hemother, 2020, Vol. 47, Page 409-419

PubMed ID: 33173459 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the cfDNA levels in the supernatants of untreated, leuko-filtered, and gamma-irradiated buffy coat PRBCs over the course of 21 days of storage and investigated the impact of hematological parameters on the change in cfDNA levels. Blood was collected from 99 blood donors and RBCs were obtained in saline-adenine-glucose mannitol (SAGM) by the buffy coat method within 2 h of collection. Buffy coats were left untreated (33 specimens), leuko-filtered (32 specimen), or exposed to 25 Gy of irradiation within 1 h of preparation (34 specimens). RBCs were centrifuged at 1,050 rpm for 9 min at 4°C after 0, 7, and 21 days of refrigerated storage and a sample of supernatant was removed. The supernatant was centrifuged at 3,000 rpm for 5 min at 4°C to remove residual cells/debris and then frozen at -80°C. WBCs, platelets, and RBC counts as well as hemoglobin and hematocrit levels were measured using an automated cell counter with the exception of the WBC count in the leuko-filtered group which was counted manually. cfDNA was extracted using the QIAamp DNA Blood Mini Kit and quantified using the Qubit dsDNA HS Assay Kit.

   Summary of Findings:

   The leuko-filtered PRBC supernatants had significantly fewer RBCs and WBCs and lower hemoglobin levels, PRBC volume, and supernatant volume than in the untreated PRBCs or the gamma-irradiated PRBCs (P<0.001, all). The leuko-filtered PRBC supernatants had higher platelet counts than the other groups but only the difference from the gamma-irradiated PRBCs was significant (P=0.024). cfDNA levels of the supernatant increased with storage of untreated PRBCs and gamma-irradiated PRBCs with significant differences noted between Day 0 and Day 7 or Day 21 and between Day 7 and Day 21 (P<0.01 all). In contrast, storage of leuko-filtered PRBCs resulted in a decline in cfDNA levels in the supernatant between Days 0 and 7 (P=0.032) but no differences were noted between Day 21 and Day 0 or Day 7. The change in cfDNA levels with storage in untreated PRBCs and gamma-irradiated PRBCs was comparable and significantly greater than that in the leuko-filtered PRBCs (P<0.001). The change in cfDNA was lower during the first 7 days than between Day 7 and Day 21 but the difference was only significant in the untreated PRBCs and gamma-irradiated PRBCs (P<0.001, both). cfDNA levels was not correlated with any of the hematological parameters but the change in cfDNA levels with storage was correlated with RBC (r=0.446, P<0.001) and supernatant volume (r=0.223, P=0.002); RBC (r=0.321, P<0.001), platelet (r=-0.374, P=0.001), and WBC counts (r=0.498, P<0.001); and hemoglobin levels (r=0.403, P<0.001).

   Biospecimens

   • Cell - Red Blood Cells

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light microscopy
   DNA	Fluorometry
   Cell count/volume	Hematology/ auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 days 7 days 21 days
   Biospecimen Aliquots and Components	Blood processing method	Leuko-filtered Gamma-irradiated Not leuko-filtered or gamma irradiated

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