Related Experiment Video
Updated: Jun 13, 2025
A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation
Published on: May 18, 2013
Preanalytical Impact of Incomplete K2EDTA Blood Tube Filling in Molecular Biology Testing
Marco Benati1, Laura Pighi1, Elisa Paviati1
1Section of Clinical Biochemistry, Department of Engineering for Innovative Medicine (DIMI), University of Verona, Piazzale L.A. Scuro 10, 37134 Verona, Italy.
Background And Aims:
The aim of this study was to investigate the possible preanalytical effect of incomplete filling of blood tubes on molecular biology assays.
Materials And Methods:
The study population consisted of 13 healthy volunteers from whom 11 mL of whole blood was collected and then distributed in different volumes (1.5, 3.0, and 6.0 mL, respectively) into three 6.0 mL spray-dried and evacuated K2EDTA blood tubes. Automated RNA extraction was performed using the Maxwell® CSC RNA Blood Kit. DNA was extracted with a MagCorePlusII, with concomitant measurement of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) gene expression. The nucleic acid concentration was calculated using the NanoDrop 1000 spectrophotometer, and purity was assessed using A260/280 and A260/230 absorbance ratios.
Results:
The RNA concentration was higher in the tubes filled with 1.5 and 3.0 mL of blood than in the reference 6 mL filled tube. The RNA 260/280 and RNA 260/230 ratios did not differ significantly between the differently filled blood tubes. The DNA concentration remained constant in the differently filled tubes. Compared to the 6.0 mL reference filled tube, the 1.5 mL and 3.0 mL filled blood tubes displayed a lower DNA 260/280 nm ratio. The DNA 260/230 ratio did not differ significantly in any of the variously filled tubes. Compared to the 6.0 mL reference filled blood tube, the 1.5 mL and 3.0 mL filled blood tubes showed a significant increase in the GAPDHcycle threshold.
Conclusions:
Our results suggest that underfilling of K2EDTA blood tubes may be a modest but analytically significant source of bias in molecular biology testing.
More Related Videos
05:58Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format
Published on: August 20, 2018
07:40Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis
Published on: March 20, 2021
Related Concept Videos
01:18Effects of EDTA on End-Point Detection Methods
In the visual method, metal-ion indicators (metallochromic dyes), which have distinct colors in their free and complex forms, are added to the mixture to signal the titration's end point. They form stable complexes with metal ions, but these complexes are weaker than the corresponding metal–EDTA complexes. As a...
01:30EDTA: Direct, Back-, and Displacement Titration
Direct titration involves buffering the metal ion solution to the desired pH and directly titrating with standard EDTA until the endpoint. The optimum pH ensures a large conditional formation constant of metal−EDTA and visibility of the free indicator color in the solution. In addition, auxiliary complexing reagents are used to prevent the precipitation of metal hydroxides...