Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

Author(s): Caboux E, Lallemand C, Ferro G, Hémon B, Mendy M, Biessy C, Sims M, Wareham N, Britten A, Boland A, Hutchinson A, Siddiq A, Vineis P, Riboli E, Romieu I, Rinaldi S, Gunter MJ, Peeters PH, van der Schouw YT, Travis R, Bueno-de-Mesquita HB, Canzian F, Sánchez MJ, Skeie G, Olsen KS, Lund E, Bilbao R, Sala N, Barricarte A, Palli D, Navarro C, Panico S, Redondo ML, Polidoro S, Dossus L, Boutron-Ruault MC, Clavel-Chapelon F, Trichopoulou A, Trichopoulos D, Lagiou P, Boeing H, Fisher E, Tumino R, Agnoli C, Hainaut P

Publication: PLoS One, 2012, Vol. 7, Page e39821

PubMed ID: 22808065 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of patient characteristics (age, BMI, gender, smoking status), timing of blood collection relative to cancer diagnosis, processing delays and extraction technique on the DNA yield from frozen buffy coat specimens. The study used a cohort of 52,256 blood specimens from 19 centers throughout Europe representing many different sub-studies on disease (cancer, heart disease, diabetes) genetics. Specimens were collected into monovette tubes containing anticoagulant (unspecified), and buffy coats were obtained by centrifugation. To each 2 mL aliquot of buffy coat, 0.5 mL of physiological solution was added, and the specimens were split and stored in straws which were stored frozen for an unspecified duration until DNA extraction.

   Summary of Findings:

   Significantly more DNA was obtained when specimens were stored in 2 straws rather than just 1 (p<0.01), from females than males (p<0.01), from smokers than non-smokers (p<0.01), using the automated LS1 than the LS2 or manual extraction (p<0.01, both) and from specimens without visible hemolysis than those with hemolysis (p<0.01). Further, the DNA yield was higher when quantified using Picogreen than Nanodrop (p<0.01), and the authors report this difference was larger when DNA was obtained from 1 straw rather than 2. More DNA was obtained from individuals who had cancer than those without (p<0.01), but there was no associated effect of cancer type. However, there was no effect of having previously been diagnosed with cancer or being diagnosed after the blood on DNA yield. DNA yield decreased with older patient age (p<0.01) and higher levels of specimen hemolysis (p<0.01), lower BMI (p<0.01), longer time between blood draw and refrigeration (p=0.02) and longer time between refrigeration and centrifugation (p<0.02). However, DNA yield was not affected by the time from centrifugation to freezing. DNA yield varied significantly between the 19 centers (p<0.01), and this variance accounted for 16.9% of the total variance in DNA yield. Importantly, insufficient DNA yield or concentration was the main reason specimens could not be used further for genotyping.

   Biospecimens

   • Bodily Fluid - Buffy Coat
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Diabetes Type 2
   • Neoplastic - Carcinoma
   • Neoplastic - Lymphoma
   • Neoplastic
   • Cardiovascular Disease

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Preaquisition	Patient age	<45 years 45-49 years 50-54 years 55-59 years 60-64 years >65 years
   Preaquisition	Patient body mass index	<25 25-27.5 27.5-30 30-35 >35
   Preaquisition	Other drugs	Former smoker Smoker Non-smoker
   Biospecimen Aliquots and Components	Aliquot size/volume	1 straw 2 straws
   Biospecimen Aliquots and Components	Hemolysis	Absent Present
   Preaquisition	Diagnosis/ patient condition	No diagnosis of cancer Current cancer diagnosis Prior cancer diagnosis Future cancer diagnosis
   Storage	Storage duration	<5 min 5 min-1 h 1-3 h > 3 h <1.5 h 1.5-2 h 2-6 h >6 h <45 min 45-59 min 1-2 h >2 h
   Analyte Extraction and Purification	Analyte isolation method	Autopure LS1 DNA extraction system Autopure LS2 DNA extraction system Manual extraction
   Spectrophotometry Specific	Technology platform	Nanodrop Picogreen
   Biospecimen Acquisition	Locale of biospecimen collection	19 centers throughout Europe
   Biospecimen Acquisition	Time of biospecimen collection	After cancer diagnosis Prior to cancer diagnosis

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