1

Ann. N.Y. Acad. Sci. 1022: 1-8 (2004). 2004 New York Academy of Sciences.
doi: 10.1196/annals.1318.002
Circulating Nucleic Acids and Proteomics of
Plasma/Serum
Clinical Utility
BRET TABACK AND DAVE S. B. HOON
Department of Molecular Oncologv. John Wavne Cancer Institute.
Santa Monica. California 90404. USA
ABSTRACT: Circulating tumor-specific nucleic acids have been identified in
plasma. serum. and other body fluids from cancer patients with tumors origi-
nating in almost any organ site. Polymerase chain reaction provides a highly
sensitive and specific technique for the detection of these genetic changes in a
limited amount of tissue/fluid. The presence of elevated levels of free DNA/
RNA in many medical conditions. malignancy. and infectious processes is being
investigated for screening. diagnosis. prognosis. surveillance for occult disease
progression. identifying potential therapeutic targets. and monitoring treat-
ment response. Additionally. elevated fetal DNA/RNA in maternal blood is
being used to determine gender identity. assess chromosomal abnormalities.
and monitor pregnancy-associated complications. Questions remain on the
etiology. characteristics. stability. and potential pathologic consequences of
cell-free DNA/RNA in the circulation. Nevertheless. nucleic acid-based assays
that monitor plasma. serum. and body fluids provide a noninvasive. facile. and
practical method for assessing patients. Proteomic profiling may prove comple-
mentary to a total functionality approach in providing a comprehensive evalu-
ation of the patient`s disease.
KEYWORDS: circulating nucleic acid; cancer; serum; plasma; proteomics
BACKGROUND
The Third International ConIerence on Circulating Nucleic Acids in Plasma/
Serum and Serum Proteomics (CNAPS III) was recently held in Santa Monica, Cal-
iIornia in coniunction with the Department oI Molecular Oncology at the John
Wayne Cancer Institute. The aim oI this conIerence was to present the latest advanc-
es in the Iield assessing circulating DNA/RNA in the areas oI cancer, viral, and pre-
natal diagnosis. Newer approaches assessing serum proteomics were also presented
that have similar potential applications. Finally, novel applications Ior the evaluation
oI circulating nucleic acids in patients with various medical conditions including
diabetes, stroke, trauma, and critical care settings were proposed. The highly suc-
cessIul nature oI this conIerence reIlects rapid growth and potential Ior clinical
Address Ior correspondence: Dr. Dave S.B. Hoon, Department oI Molecular Oncology, John
Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404, USA.
hoon(iwci.org
2 ANNALS NEW YORK ACADEMY OF SCIENCES
translation. In addition, technological advancements Ior nucleic acid and protein
detection Irom blood along with high through-put automated systems Ior specimen
processing and analysis will yield readily available qualitative results with the goal
oI improving decisions in patient care.
The Iield oI CNAPS was Iirst believed to originate in 1947 when Mandel and
Metais
1
described the successIul isolation oI DNA Irom plasma. However, it was not
until the 1970s when newer techniques were developed to detect submicrogram lev-
els oI DNA did potential application in cancer become apparent.
2,3
During the
approximate 30-year interim, studies oI circulating DNA were restricted mostly to
those states related to autoimmunity, such as systemic lupus erythematosus and
rheumatoid arthritis, where levels in blood were highly abundant.
47
Despite grow-
ing evidence, skepticism remained until Stroun and Anker
8,9
elucidated the etiology
oI circulating Iree DNA as tumor-derived, which provided the impetus Ior successIul
development oI this Iield. Subsequently, an increasing number oI publications have
demonstrated the presence oI circulating tumor-associated Iree nucleic acids in pa-
tients with various types oI cancer. In addition, circulating Ietal DNA in maternal
blood has been investigated as a potentially valuable noninvasive source Ior Ietal
sexing, assessing chromosome ploidy, and monitoring pregnancy.
10,11
The Iollow-
ing is a summation oI the highlights Irom the Third International ConIerence on
Circulating Nucleic Acids in Plasma/Serum and Proteomics.
DNA
Cancer development is associated with the progressive accumulation oI genetic
and epigenetic alterations. These events can include allelic loss, microsatellite insta-
bility, point mutations, ampliIication, translocation, and gene promoter region
hypermethylation. IdentiIication oI these genetic changes detected in the circulation
has been described Ior almost every organ system in cancer patients. These circulat-
ing nucleic acids identiIied in plasma/serum provide a potential role as molecular
markers Ior assessing patients` prognosis, predicting and monitoring response to
therapy, and identiIying the burden oI occult residual disease.
Loss oI heterozygosity (LOH) is one oI the most Irequently described genetic
alterations associated with solid tumors. The same genetic alterations have been
identiIied in the plasma and serum oI patients with cancer oI the breast, lung, head
and neck, gastrointestinal tract, and urologic and gynecologic organs and in mela-
noma.
1219
Microsatellite analysis Ior LOH can be perIormed with commercially
available short tandem repeat (STR) markers that map to every chromosome and are
highly inIormative. These markers are labeled with radioactive or Iluorescent dyes,
assayed by gel electrophoresis or high through-put capillary arrays.
Hoon`s group demonstrated applications Ior assessing patients with melanoma.
They showed that the presence oI circulating microsatellites Ior LOH could predict
a lack oI response to biochemotherapy in patients with advanced metastatic melano-
ma. More common malignancies, those oI the breast and prostate, were shown to
have circulating LOH in 50 oI cases by Schwarzenbach et al. Furthermore, Taback
demonstrated that the presence oI circulating microsatellite markers Ior LOH could
be detected in bone marrow, which may provide an additional site, with blood, Ior
assessment. Using a 17 microsatellite marker panel, corresponding to 7 chromo-
3 TABACK & HOON: CIRCULATING NUCLEIC ACIDS
somes Irequent Ior LOH, von Knobloch`s group was able to detect urinary tract can-
cers with a speciIicity and sensitivity oI 80. Demonstrating that microsatellite
alterations could be identiIied Irom other body Iluid sites, Burns Iound LOH in se-
rum and peritoneal washings Irom ovarian cancer patients with early stage disease,
which may provide a sensitive/speciIic marker Ior Iollow-up. Additionally, Fleisch-
hacker et al. detected microsatellite alterations in bronchial alveolar lavage Iluid,
which proved more sensitive than cytology in a pilot evaluation oI a small group oI
patients with lung cancer. For widespread acceptance oI this technique, standards are
still needed that are uniIorm in the deIinition oI LOH. Methods that decrease con-
taminating normal DNA may Iurther improve sensitivity oI the assay. Sidransky
demonstrated eIIorts Ior assessing circulating methylation markers as a diagnostic
aid to conventional medical imaging modalities Ior cancer screening in high risk
patients. Furthermore, they have identiIied circulating mitochondrial DNA muta-
tions in cancer patient`s body Iluids and suggest that because oI the homoplasmic
nature oI mitochondrial DNA it may prove more sensitive than genomic DNA as a
cancer marker. Other alternatives include quantitative assessments oI DNA. Swam-
inathan et al. presented an approach using genome equivalents in plasma to diIIer-
entiate patients with normal, benign prostatic hypertrophy, prostate intraepithelial
neoplasia, and invasive cancer. Similarly, this group has associated increased plasma
DNA in intensive care unit patients with iniury severity scores and overall outcome.
0XWDWLRQV
Point mutations provide another suitable target Ior plasma/serum analysis. A
common point mutation is K-ras oncogene. This mutation occurs in most pancreatic
cancers (95) and in many colorectal cancers (50) and lung cancers (33).
20
Its
high Irequency in select cancers, unique association with malignancy when detected,
and common presentation at codon 12 makes K-ras a very enticing blood tumor
marker Ior investigation. Assays oI tumor specimens Ior mutation are highly speciIic
and sensitive. Kimura`s group Iound a 20 incidence oI K-ras mutation in the plas-
ma oI patients with non-small cell lung cancer on a phase I clinical trial, proving that
detection is Iacile in the clinical setting. Fleischhacker and colleagues successIully
identiIied TP53 mutations in plasma Irom patients with gastrointestinal tumors and
Iound resolution in some patients aIter treatment. These Iindings suggest that circu-
lating tumor-related nucleic acids may prove promising Ior monitoring patient`s
response to therapy. Alternatively, Gormally`s group studied the predictive value oI
cancer-related mutations in plasma DNA oI healthy volunteers with the purpose oI
clariIying the relation between air pollution, tobacco or environmental Iactors, and
the subsequent development oI cancer. Using K-ras as a screening tool, they showed
it preceded the development oI cancer in a small group oI patients, and it was asso-
ciated with the presence oI bulky DNA adducts. The inability to identiIy genetic
alterations in plasma/serum in all patients with cancer may exist, in part, because not
all tumors shed DNA into the blood, and/or DNA Iragments containing the mutation
may have limited stability in the circulation. Furthermore, the presence oI circulat-
ing normal DNA may interIere with the assay`s sensitivity.
Recently, BRAF mutation was described in approximately two thirds oI cutane-
ous melanomas and papillary thyroid cancers, providing an attractive tumor-speciIic
marker Ior assessment.
21,22
Studies demonstrating the presence oI BRAF mutation
4 ANNALS NEW YORK ACADEMY OF SCIENCES
in serum Irom patients with melanoma and its potential clinical utility were present-
ed by Hoon`s group. A highly sensitive mutation-scanning platIorm to assess BRAF,
K-ras, APC, and TP-53 gene mutations in plasma samples Irom patients with colo-
rectal cancer was demonstrated by Lilleberg and Transgenomic Inc. TrimGen Cor-
poration revealed their cost-eIIective, Iacile mutation detection assay that is based
on a colorimetric shiIt-termination technique. Other corporate sponsors were in
attendance at the conIerence to present additional novel technologies that enhance
nucleic acid detection in plasma/serum and body Iluids. Xenomics Inc. demonstrat-
ed technology using Stencil-Aided Mutation Analysis (SAMA) to reduce wild-type
sequences, and they successIully identiIied mutant K-ras DNA Irom 0.1 mL oI urine
in 10 oI 15 patients with primers designed Ior 87-bp amplicons. A microIluidic 'lab
on a chip was presented by Caliper Technologies, which integrates reaction assem-
bly and thermocycling to increase signal-to-noise ratios that detect 1 variant amonga
background oI 700 wild-type sequences. Rubicon Genomics Inc. presented an assay
that integrates whole genome, transcriptome, and methylome into a single platIorm
to ampliIy whole spectrum oI sequences >1,000-Iold.
+\SHUPHWK\ODWLRQ
The epigenetic phenomenon oI promoter region hypermethylation in many solid
tumors is another gene-silencing aberration that may contribute to cancer develop-
ment.
23
Methylation-speciIic polymerase chain reaction (PCR) is an extremely sen-
sitive and Iacile assay Ior the detection oI occult circulating tumor cells and tumor-
associated DNA in the blood and body Iluids oI cancer patients.
24
Herman`s group
demonstrated variations in the hypermethylation proIile between sporadic and
hereditary Iorms oI breast and colon cancers. In addition, gene hypermethylation
analysis can divide tumors into a hierarchical cluster. These Iindings provide hyper-
methylation gene targets Ior assessing a cancer patient`s disease.
Mller et al. identiIied hypermethylation oI RASSF1A and/or APC Irom an
initial panel oI 39 genes that, when detected in the blood oI 62 breast cancer patients,
was an independent predictor oI decreased overall survival. Cairns showed that
assessment oI urine using a hypermethylation panel consisting oI six genes correlat-
ed with urinary tract tumors with 100 speciIicity and 80 sensitivity. However,
because hypermethylation is not a Iixed event, it may not always be an accurate/
reliable tumor marker. Variable gene methylation patterns between diIIerent normal
tissues can Iurther limit an assay`s speciIicity. Technologies that improve methods
Ior DNA collection and enhance enrichment Ior the marker(s) oI interest and the
addition oI nested or real-time PCR may Iacilitate gene detection.
NUCLEOSOMES
Another exciting area oI research involving nucleosomes was addressed at the
conIerence. Holdenreider et al. identiIied circulating nucleosomes in the serum oI
cancer and stroke patients. His group discussed methods Ior detection including the
use oI a sandwich antibody to DNA and histone protein complexes, serum kinetics
Iollowing radio/chemotherapy, and potential application Ior monitoring cancer
patient`s treatment response. These Iindings present a new approach Ior assessing
circulating nucleic acids in plasma and serum.
5 TABACK & HOON: CIRCULATING NUCLEIC ACIDS
VIRAL DNA
Viral-associated DNA that contributes to cellular transIormation and tumorigen-
esis provides an attractive plasma/serum marker because it is highly speciIic, oIten
circulates in great abundance, and is easily distinguished Irom normal host DNA.
The Epstein-Barr viral (EBV) DNA marker Ior nasopharyngeal carcinoma is the
most thoroughly investigated DNA tumor marker to date and holds the greatest
promise Ior clinical application.
25,26
Plasma/serum kinetics oI Ireely circulating
EBV DNA has been well-characterized in patients with nasopharyngeal cancers Iol-
lowing radiation treatment.
27
Recently, Lo et al. assessed circulating EBV DNA in
patients aIter surgery (nasopharyngectomy), again demonstrating Iirst-order kinet-
ics; however, clearance appeared more quickly with surgery than with radiotherapy
(median halI-liIe: 2 hr and 20 min versus 3.8 days). In addition, the same investiga-
tors are assessing the nature oI circulating EBV DNA and have demonstrated in
well-constructed experiments that it is present in the Iorm oI true DNA Iragments
rather than virions. Ngan identiIied EBV DNA in serum oI patients with lympho-
proliIerative type lung cancer and showed elevated levels to correlate with the pres-
ence oI disease. Hacker et al. evaluated levels oI circulating hepatitis B viral DNA
and RNA and their characteristics in response to Lamivudine drug treatment. UnIor-
tunately, to date, Iew human malignancies are so strongly correlated with viral
oncogenes.
FETAL DNA
IdentiIication oI nucleated Ietal cells in maternal circulation is a noninvasive
technique Ior prenatal diagnosis.
28
However, despite transplacental migration oI
Ietal cells, only rarely are they identiIied in the maternal circulation.
29
By contrast,
cell-Iree Ietal DNA can be detected in greater abundance in maternal plasma and
serum and as early as the sixth week oI gestation.
30
Concentrations increase
throughout each trimester oI pregnancy, with the greatest rise iust prior to parturi-
tion.
31
The use oI PCR to locate Y chromosomal sequences in the plasma/serum can
identiIy more than 70 oI women carrying male Ietuses.
11
Bianchi is evaluating
circulating Ietal DNA as a noninvasive assessment tool Ior Ietal molecular karyo-
typing and determination oI abnormal ploidy. In addition, this group is perIorming
quantitative assessments oI Ietal DNA, which may provide a more sensitive method
to monitor alterations in pregnancy such as preeclampsia and preterm labor.
32,33
Chiu is adapting technologies Ior the detection oI single nucleotide polymorphisms
(SNPs) and methylation to the evaluation oI Ietal DNA.
Issues still to be addressed include identiIication oI markers Ior Iemale Ietuses,
better methods Ior enrichment oI Ietal DNA, and techniques to limit contamination
Irom maternal DNA species. Because the origin oI this circulating Ietal DNA re-
mains relatively unknown, any potential pathophysiologic impact on the maternal
host is ill-deIined. These Iindings suggest that circulating Ietal DNA in maternal
blood may present a unique source oI genetic material Ior prenatal noninvasive di-
agnosis oI genetic diseases, assessment oI abnormal chromosome numbers, determi-
nation oI gender, and monitoring pregnancy-associated complications.
6 ANNALS NEW YORK ACADEMY OF SCIENCES
SERUM PROTEOMICS
Because the potential applications oI serum proteomics share many oI those Ior
CNAPS, it has been included in this conIerence Ior the Iirst time. Discussions
included protein chip surIace-enhanced laser desorption ionization (SELDI) and
matrix-assisted laser desorption ionization time oI Ilight (MALDI-TOF) prepara-
tion, processing, and clinical utilities. Wilson and Sidransky demonstrated represen-
tative examples oI serum proteomic proIiling Ior assessing cancer patients, whereas
Gerton is evaluating its role in the diagnosis oI ectopic pregnancy. Chan reviewed
the systematic process Ior biomarker discovery Irom protein proIiles to identiIica-
tion to validation. Johann discussed the concerted eIIorts oI the National Institutes
oI Health, National Cancer Institute, and the Food and Drug Administration to
develop platIorm technologies in proteomics that identiIy unique protein proIiles Ior
early disease detection and interpretive pathology, elucidating potential targets in
cell signal transduction pathways, tailoring individual patient therapies, and moni-
toring treatment response. Some limitations associated with proteomics must still be
addressed including its variability and imprecision, lack oI quantitation, tendency
Ior its results to reIlect high abundance proteins, and bioinIormatical analysis
procedures.
Srivastava provided an overview oI the challenges associated with biomarker dis-
covery and the role oI the NCI, Early Detection Research Network (EDRN). Chan
presented the role oI FDA regulation in in vitro diagnostics. In addition. historical
aspects oI medical device regulations, classiIication oI medical devices, the submis-
sion pathway, and maior elements Ior submission were reviewed in detail.
CONCLUSIONS
Circulating nucleic acids have been described in the plasma, serum, and body
Iluids oI patients with a variety oI cancers. This cell-Iree tumor-associated DNA may
contain allelic losses, microsatellite instability, mutations, ampliIications, promoter
region hypermethylation, and/or viral-associated elements. Likewise, Ietal DNA has
been documented in the blood oI women during pregnancy. Assessment oI circulat-
ing nucleic acids in plasma and serum oIIers the potential Ior noninvasive screening,
diagnosis, prognosis, measuring response to treatment, and evaluating disease
pathophysiology and biology. In contrast to Ietal and tumor cells, which may remain
in circulation Ior extended periods, Ietal and tumor-associated DNA appears to be
rapidly cleared according to Iirst-order kinetics.
27,34,35
Furthermore, blood levels oI
Ietal and tumor-related Iree DNA appear to be higher than levels oI circulating Ietal
and cancer cells, respectively, and can readily be detected by current PCR tech-
niques.
31,36,37
Thus, DNA-based assays may prove clinically inIormative and corre-
late closely with disease outcome.
Because nucleic acids can be identiIied in plasma, serum, bone marrow aspirates,
urine, prostatic Iluid, peritoneal Iluid, amniotic Iluid, cerebrospinal Iluid, bronchial
lavage, gastric and biliary iuices, and stool, their evaluation may serve as a useIul
tool to assess patients with cancer, inIectious diseases, various medical conditions,
organ transplants, trauma, and critical illnesses. Questions persist regarding the true
nature oI circulating DNA/RNA, its etiology, its stability/halI-liIe in various body
7 TABACK & HOON: CIRCULATING NUCLEIC ACIDS
Iluids, and its possible pathogenicity. When these issues are resolved, the evaluation
oI circulating nucleic acids in coniunction with gene transcription and proteomic
proIiling will become a cornerstone oI an integrative Iunctional 'omics approach to
the comprehensive assessment oI disease. Ongoing technological advancements that
improve methods Ior nucleic acid isolation, detection, and analysis will lead to more
automated approaches Ior rapid and accurate processing oI multiple specimens.
These highly sensitive and speciIic assays should generate readily reproducible
quantitative and qualitative results in a cost-eIIective manner that will improve
patient care.
The CNAPS III ConIerence brought together individuals Irom a number oI coun-
tries representing a variety oI disciplines. It was apparent Irom the large attendance
at this conIerence and the accompanying integrated workshop that the Iield has
moved rapidly Iorward with potential application Ior many new diseases. Irrespec-
tive oI the diverse interests present, there was a united eIIort to continue our under-
standing and determine the clinical utility oI circulating nucleic acids and
proteomics in plasma/serum Ior medical conditions.
ACKNOWLEDGMENTS
This work was supported in part by the CaliIornia Breast Cancer Research Pro-
gram (Grant 7WB-0021), Department oI DeIense (DAMD17-01-1-0279), National
Cancer Institute (Grants P01CA29605 and R21CA100314), the Leslie and Susan
Gonda (Goldschmied) Foundation (Los Angeles), the Rachel Goodman Cancer
Research Grant, and the Fashion Footwear Association oI New York.
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