The Importance of Careful BloodProcessing in Isolation of Cell-Free DNA

KAREN PAGE,a TOM POWLES,b MARTIN J. SLADE,b MANUELATAMBURO DE BELLA,b ROSEMARY A. WALKER,a R. CHARLESCOOMBES,b AND JACQUELINE A. SHAWa

aDepartment of Cancer Studies and Molecular Medicine, University of Leicester,Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary,Leicester LE2 7LX, UKbDepartment of Cancer Medicine, Hammersmith Campus, Imperial CollegeSchool of Medicine, MRC Cyclotron Building, Du Cane Road, London W12 0NN,UK

ABSTRACT: In healthy individuals, the source of cell-free plasma DNAis predominantly apoptotic, whereas, increased plasma DNA integrity isseen in cancer patients. Therefore, it is important to carefully isolate ab-solutely “cell-free” plasma DNA. Plasma DNA from 30 healthy femaleswas analyzed using 4 PCR amplicons of increasing size, comparing stan-dard blood processing with additional centrifugation steps prior to DNAextraction. Cellular DNA contamination, indicated by positive ampli-cons >300 bp was eliminated only after the extra centrifugation step.This highlights the importance of careful processing in preparation ofcell-free plasma DNA as a tool for cancer detection and we recommendthe use of a microcentrifuge spin, prior to DNA extraction.

KEYWORDS: cell-free plasma DNA; PCR; apoptosis

INTRODUCTION

The presence of free-circulating nucleic acid in the blood has been recog-nized since the 1970s,1 with increasing quantities found in cancer patients.2

The identification of DNA exhibiting tumor-specific LOH in plasma from pa-tients with lung3 and head and neck cancer4 respectively, prompted a widerinvestigation of other cancer types. For breast cancer patients, we5 and others6

have shown that tumor DNA in plasma at diagnosis may be a valuable predictor

Address for correspondence: Dr. Karen Page, Department of Cancer Studies and Molecular Medicine,Robert Kilpatrick Clinical Sciences Building, University of Leicester, Leicester, LE2 7LX, UnitedKingdom. Voice: +44-116-252-3239; fax: +44-116-252-3274.

e-mail: kd18@leicester.ac.uk

Ann. N.Y. Acad. Sci. 1075: 313–317 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1368.042

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of disease-free survival. However, in order to develop novel methods of cancerdetection using analysis and quantitation of plasma DNA, it is important tocontrol for contamination by cellular DNA, which might interfere with theanalysis. In healthy individuals the source of cell-free plasma DNA is pre-dominantly apoptopic7 and so only small DNA fragments (<300 bp) shouldbe detected. Therefore, we have used PCR analysis of increasing-sized ampli-cons to analyze plasma DNA isolated from normal, healthy female donors toinvestigate the utility of standard blood processing for successful isolation ofplasma DNA. We have also compared the use of an additional centrifugationstep both before and after storage of plasma on the recovery of cell-free DNA.Recently, Chiu et al. have recommended an extra centrifugal step, in additionto initial plasma separation, to produce absolutely cell-free DNA.8

MATERIALS AND METHODS

Blood Collection, Processing, and DNA Extraction

Twenty milliliters venous blood samples were withdrawn from a peripheralvein into EDTA-containing tubes from three groups of normal healthy femaledonors (n = 30). Following plasma separation by centrifugation at 2000 rpmfor 10 min (×2), the first two sets of samples were stored directly at −80◦C.The third set was subjected to an extra spin, 13,500 rpm for 5 min in a bench-topmicrofuge and the supernatant was removed to a clean Eppendorf tube prior tostorage. After thawing of plasma, all samples were split into two aliquots. Thefirst was subjected to no further treatment and the second to centrifugation at13,500 rpm for 5 min in a bench-top microfuge. Plasma DNA was extractedfrom all samples using the QiAamp Blood Kit (Qiagen, Hilden, Germany)according to the blood and body fluid protocol, modifying the initial plasmasample (500 �L) and final elution (100 �L) volume.

PCR Analysis

DNA recovered from stored plasma was analyzed by PCR using five am-plicons of increasing size (100 bp and 350 bp GAPDH and 512 bp, 843 bp,1271 bp Bcl-2 amplicons). Primer details are given in TABLE 1. Ten microlitersof each plasma DNA sample was analyzed by PCR as described previously5

using the following cycles: 98◦C for 3 min, followed by 35 cycles of 94◦Cfor 30 sec, 60◦C for 30 sec, and 72◦C for 30 sec to 2 min depending onthe amplicon length, with a final extension of 7 min at 72◦C. All reactionswere carried out on a GeneAmp PCR System 9700 (Perkin–Elmer AppliedBiosystems, Warrington, UK). PCR products were resolved on 1–4% agarosegels.

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TABLE 1. PCR primers for 100 bp and 350 bp GAPDH and 512 bp, 843 bp, 1271 bp Bcl-2amplicons

Primer/amplicon Sequence

GAPDHF (100 bp) GCAAGAGCACAAGAGGAAGAGAPDHR (100 bp) ACTGTGAGGAGGGGAGATTC

GAPDH (350 bp) Given in Hall et al.9

Bcl-2 RP1(common) ATAGCAGCACAGGATTGGBcl-2 FP2 (512 bp) GTGCATTTCCACGTCAACBcl-2 FP3 (843 bp) GATGGAATAACTCTGTGGCBcl-2 FP4 (1271 bp) AGAAGGACATGGTGAAGG

RESULTS

When we compared the effect of different processing steps prior to DNAextraction on the range of amplicons detected following PCR of plasma DNA,we found a wide variation between the three sets of samples. With no ex-tra processing, all samples from sets 1 and 2 showed amplification of thetwo smallest amplicons, and several samples also showed amplification ofthe larger Bcl-2 amplicons (FIG. 1). However, for plasma DNA from sets 1

FIGURE 1. PCR analysis of plasma DNA from (A) control set 1 (n = 11) and (B)control set 2 (n = 11) with no additional centrifugation. Analysis of 100 bp and 350 bpGAPDH and 512 bp, 843 bp, 1271 bp Bcl-2 amplicons. Bl = water blank.

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FIGURE 2. Analysis of plasma DNA from (A) control set 1 and (B) control set 2 withadditional 5-min microfuge spin prior to DNA extraction. Analysis of 100 bp and 350 bpGAPDH and 512 bp, 843 bp Bcl-2 amplicons. Bl = water blank.

FIGURE 3. Analysis of plasma DNA from control set 3 (n = 7) with additional 5-minmicrofuge spin prior to storage and DNA extraction. Analysis of 100 bp and 350 bp GAPDHand 512 bp, 843 bp Bcl-2 amplicons. Bl = water blank.

and 2 recovered after the additional 5-min microfuge spin, none of the threelargest amplicons were detected (control sets 1 and 2, FIG. 2). Set 3, which hadbeen subjected to a 5-min spin in a bench-top microfuge prior to storage andDNA extraction, only showed amplification of the smallest 100-bp amplicon(FIG. 3). In comparison, in our routine analyses of plasma DNA isolated frombreast cancer patients, with the addition of a microfuge spin prior to DNAextraction, most plasma DNA samples successfully amplify large amplicons(>500 bp in size) (data not shown).

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DISCUSSION

Recently, it has been shown that additional centrifugation may be requiredto eliminate contaminating cellular DNA from plasma DNA samples.8 Weexamined whether additional microfuge centrifugation would eliminate con-taminating cellular DNA from “normal” plasma samples, isolated from healthyfemales (age range 25–55 years), using PCR analysis of a number of increasing-sized amplicons. Cellular DNA contamination was indicated by successful am-plification of products >300 bp in size, evident in control sets 1 and 2 (FIG. 1),which then decreased or disappeared after additional microfuge centrifugation(FIG. 2).

Since healthy individuals have very low levels of free-circulating DNA, theamplified DNA observed prior to additional centrifugation was most likelyderived from contaminating cellular DNA. The addition of a 5-min spin in abench-top microfuge prior to storage and or DNA extraction from plasma, wassufficient to eliminate contaminating cellular DNA, as revealed by the absenceof amplification of products >300 bp in length (control set 3, FIG. 3).

Since plasma DNA is a promising biomarker for cancer detection and in-creased tumor DNA integrity in plasma is associated with cancer,7 it is vitalto process blood samples appropriately in order to minimize contaminationby cellular DNA. Our data highlight the importance of careful processing inpreparation of cell-free plasma DNA for cancer detection and we recommendthe use of a high-speed spin in a bench to microcentrifuge, prior to storageand/or DNA extraction.

REFERENCES

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2. KOFFLER, D. et al. 1973. The occurrence of single stranded DNA in the serum ofpatients with SLE and other diseases. J. Clin. Invest. 52: 198–204.

3. CHEN, X.Q. et al. 1996. Microsatellite alterations in plasma DNA of small cell lungcancer patients. Nat. Med. 2: 1033–1035.

4. NAWROZ, H. et al. 1996. Microsatellite alterations in serum DNA of head and neckcancer patients. Nat. Med. 2: 1035–1037.

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6. SILVA, J.M. et al. 1999. Presence of tumour DNA in plasma of breast cancer patients:clinicopathological correlations. Cancer Res. 59: 3251–3256.

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9. HALL, L.L. et al. 1998. Reproducibility in the quantification of mRNA levels byRT-PCR-ELISA and RT Competitive-PCR-ELISA. Biotechniques 24: 652–658.