Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Preanalytical Impact of Incomplete K(2)EDTA Blood Tube Filling in Molecular Biology Testing.

Author(s): Benati M, Pighi L, Paviati E, Visconti S, Lippi G, Salvagno GL

Publication: Diagnostics (Basel), 2024, Vol. 14, Page

PubMed ID: 39272719 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of incomplete tube filling (25%, 50% and 100%) on the concentration and purity of RNA and DNA from K₂EDTA blood collected from healthy volunteers. Blood was collected from thirteen healthy volunteers (4 males and 9 females; 26-66 years of age) into a syringe using a 21-gauge needle. A total of 1.5 mL, 3 mL and 6 mL of blood were added to 6 mL K₂EDTA vacutainers, resulting in a 25%, 50% and 100% fill volume, respectively. RNA was extracted from 1 mL of each blood specimen using the Maxwell RSC RNA Blood Kit and DNA was extracted from an unspecified volume of blood using the MagCorePlus II Kit. RNA and DNA were quantified and their purity evaluated by spectrophotometer. RNA and DNA were further quantified by real-time PCR of ABL and GAPDH, respectively.

   Summary of Findings:

   The concentration of RNA was significantly higher in the tube with only 1.5 mL (25% fill) or 3 mL (50% fill) versus 6 mL (100% fill) of blood (62.44 ng/μL and 59.84 ng/μL versus 56.58; p=0.001 and p=0.021, respectively). The copy number of ABL was also higher in RNA extracted from the partially filled tubes than the completely filled tubes, but the difference was only significant between the tube filled with 1.5 mL (25% fill) relative to the tube with 6 mL (100%) of blood (mean 8,225 copies versus 7,343 copies, p=0.026).  While DNA concentration was not significantly affected by fill volume, the CT values for GAPDH were higher when the tube was filled with 1.5 mL (25% fill) or 3 mL (50% fill) versus 6 mL (100% fill) of blood (p=0.026 and p=0.017, respectively). RNA purity, as evaluated by the ratio of absorbance at 260/280 nm or 260/230 nm, was not affected by fill volume but DNA purities, as measured by the 260/280 ratio, was higher when extraction was from 6 mL rather than 1.5 or 3 mL of blood (mean 1.75 both versus 1.77, p=0.047 and p=0.021, respectively).

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of fluid acquisition	Properly filled tube Improperly filled tube
   Biospecimen Aliquots and Components	Aliquot size/volume	1.5 mL (25% filled) 3 mL (50% filled) 6 mL (100% filled)

   View more