Logistics and quality control for DNA sampling in large multicenter studies.
Author(s): Nederhand RJ, Droog S, Kluft C, Simoons ML, de Maat MP
Publication: J Thromb Haemost, 2003, Vol. 1, Page 987-91
PubMed ID: 12871366 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of storage and mechanical disruption on the DNA yield, quality and amplification success from blood specimens. Blood was collected from 8 healthy individuals into K3EDTA tubes, and DNA was extracted by salting-out and stored at 4 degrees C. The PCR target was not specified. Mechanical disruption was conducted on a head-over vortex for 24 h or by mailing (unspecified distance, duration and temperature).
Summary of Findings:
DNA yield decreased with increasing storage of blood at room temperature, with a 50% reduction observed by day 28 compared to day 0, but DNA yield was not affected by up to 28 days of storage at 4 or -20 degrees C. For specimens stored for >3 days, the average OD260/280 ratios were 1.7 for specimens stored at room temperature, 1.8 for specimens stored at 4 degrees C, and 1.6 for specimens stored at -20 degrees C with more variability as well, but significance was not stated. The authors report PCR was successful from all specimens stored at room temperature, 4 degrees C, and -20 degrees C. The authors report visible erythrocyte lysis increased with increasing duration of room temperature storage which resulted in a need for repeated lysis steps and increased pronase to lyse the leukocyte pellet, but less lysis was observed with storage at 4 degrees C. The authors report that exposure of the specimens to extreme conditions (head-over vortex for 24 h, or mail for 24 h) resulted in hemolysis and variable OD260/280 ratios like those observed after storage at -20 degrees C, but similar to specimens stored at -20 degrees C there was no change in DNA yields.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Electrophoresis DNA PCR DNA Spectrophotometry Cell count/volume Macroscopic observation Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 days
3 days
7 days
14 days
21 days
28 days
Storage Storage temperature -20 degrees C
4 degrees C
Room temperature
Storage Between site transportation method Mailed
Not transported
PCR Specific Length of gene fragment 200 bp
1200 bp
Biospecimen Aliquots and Components Hemolysis Not induced
Vortex-induced
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Study Purpose
The purpose of the study was to determine the effects of delayed lysis due to transport on hemolysis. This study used 11,241 specimens from a European study on the effects of perinodopril in patients with coronary artery disease. Whole blood specimens were mailed to a central laboratory in a protective box where the blood was lysed, and the leukocyte pellet was stored frozen at -20 degrees C. The delay in lysis due to storage versus transport was not specified.
Summary of Findings:
Increased time between blood collection and processing resulted in increased hemolysis, with the majority of specimens subjected to a delay of 5 days or more before lysis showing some hemolysis. In 93% of specimens, delays before lysis had no effect on leukocyte pellet size, and the authors report that, using a subset of 61 specimens, leukocyte pellet size was not associated with DNA yield or quality. Further, the authors report that all DNA obtained was of high molecular weight and without visible degradation, regardless of the duration of the processing delay.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Coronary Artery Disease
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Spectrophotometry Cell count/volume Macroscopic observation Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0-30 days
Storage Between site transportation method Mailed
Storage Specimen transport duration/condition 1-30 days