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Clinical Chemistry 49, No. 3, 2003

Althaia Xarxa Asistencial de Manresa

La Culla, s/n
08240 Manresa (Barcelona), Spain
*Author for correspondence. Fax 34-938743859; e-mail hgmlaboratorio@aehh.org.

Effects of Blood-Processing Protocols

on Cell-free DNA Quantification in
Plasma

To the Editor:
Recently, qualitative analysis of cellfree DNA in blood plasma has attracted much interest for the diagnosis of cancer, fetal gender, Rhesus D
status, and inherited disorders (1, 2 ).
Other studies have shown that quantification of total plasma DNA may
indicate fetal chromosomal aneuploidies and pregnancy-associated
complications or the presence/recurrence of cancer (1, 3, 4 ). Irrespective
of the type of study (qualitative or
quantitative DNA analysis), it is important that cell-free plasma DNA is
not contaminated with cellular DNA
that interferes with analysis and accurate quantification.
To prevent cellular DNA contamination, Chiu et al. (5 ) emphasized
the need of preanalytical standardization of blood-processing protocols. The authors note that after centrifugation of blood at a low speed
(800g), the amount of isolated DNA
from plasma is affected by the presence of cells that remain in the
plasma fraction. An additional centrifugation step (16 000g) or filtering
of the plasma is necessary to produce
absolutely cell-free plasma DNA. Because most laboratories, including
ours, centrifuge blood at relatively
low speeds (800 1500g) and store
plasma without additional treatment, these results may have a serious impact on the usefulness of previously collected plasma samples for
retrospective DNA analysis/quantification.
To confirm the results of Chiu et al.
(5 ), we collected EDTA blood in
7-mL Vacutainer Tubes from 18
healthy women with uncomplicated
pregnancies, attending the outpa-

Fig. 1. Albumin concentrations in plasma from pregnant (A) and nonpregnant (B) women.
(A), albumin concentrations (CE/mL) isolated from plasma samples obtained from 18 pregnant women,
collected in 7-mL EDTA-blood Vacutainer Tubes. The tube volume (7.0 mL) and centrifugal forces (800g and
16 000g) before plasma collection and storage at 20 C are shown on the x axes. Fraction A, median, 485
CE/mL [95% confidence interval (CI), 371207 CE/mL]; fraction B, median, 422 CE/mL (95% CI, 227-1911
CE/mL). A two-sided paired t-test showed no significant difference ( 0.27) in albumin concentrations
between fractions A and B. (B), albumin concentrations isolated from plasma samples obtained from 10
nonpregnant donors, collected in 10- and 7-mL EDTA-blood Vacutainer Tubes. High-speed centrifugation
(16 000g) occurred either before storage (fraction B) or after storage at 20 C (fraction C). Fraction A,
median, 24 936 CE/mL (95% CI, 1069 123 079 CE/mL); fraction B, median, 480 CE/mL (95% CI, 203 897
CE/mL); fraction C, median, 453 CE/mL (95% CI, 272961 CE/mL); fraction D, median, 682 CE/mL (95% CI,
216-2652 CE/mL). A two-sided paired t-test showed a significant difference ( 0.01) in albumin
concentrations between fractions A and B. Differences between other pairs of fractions (B and C, B and D, C
and D) were not significant ( 0.57, 0.25, and 0.28, respectively).

tient clinic of the University Medical

Centre Nijmegen, after 21 weeks of
gestation. Blood was centrifuged at
800g for 10 min. The plasma super-

natant was carefully removed to

0.5 cm above the buffy coat layer,
homogenized, and divided into two
portions: fractions A (400 L) and B

526

(450 L). Fraction B was centrifuged

additionally at 16 000g for 5 min, and
400 L of the supernatant was transferred to a new tube. All fractions
were processed within 3 h after collection and stored at 20 C for at
least 1 month. After thawing, the
DNA in the fractions was extracted
exactly according to the protocol of
Chiu et al. (5 ). Both fractions A and B
from each pregnant woman were
processed in the same batches during
the isolation and quantification procedures. Copies of the albumin gene
were quantified with real-time PCR
and transformed to cell-equivalents/mL of plasma (CE/mL), as described previously (3 ).
The quantification results for
plasma fractions A and B from 18
pregnant women are shown in Fig.
1A. Although the median number of
cell-equivalents in fractions B (422
CE/mL) correlated well with the results of Chiu et al. (5 ), surprisingly
we detected no significant amounts
of additional DNA (from cellular origin) in fractions A. Obviously, in our
samples, all cellular DNA had been
removed from the plasma by centrifugation at 800g. After careful reanalysis of the protocol of Chiu et al., we
could identify only a single difference with our procedure: we collected blood in 7-mL EDTA collection tubes instead of 10-mL tubes.
To demonstrate the possible effect
of this difference, we performed an
additional experiment in which we
collected blood from 10 nonpregnant
donors into both 10- and 7-mL tubes.
Blood and plasma were processed as
described before. In these samples,
after DNA isolation, contaminating
cellular DNA was still present in the
plasma fraction of the 10-mL collection tubes after centrifugation at 800g
(Fig. 1B, fraction A). It was removed

Letters

after additional centrifugation at

16 000g, confirming the results of
Chiu et al. (Fig. 1B, fraction B). Moreover, plasma DNA concentrations
derived from the plasma from 7-mL
tubes (Fig. 1B, fraction D) were identical to the DNA concentrations in
cell-free plasma (Fig. 1B, fraction B),
corroborating our previous results
shown in Fig. 1A.
From these results, we concluded
that the amount of blood collected
(i.e., the size of the blood collection
tube) affects the presence of contaminating cells in the plasma fraction.
Because these cells were present in
plasma both from pregnant women
(5 ) and nonpregnant individuals
(our results), leukocytes are the most
likely explanation for the contaminating cells in the 10-mL blood collection tubes.
In parallel, we questioned whether
these contaminating cells in plasma
(Fig. 1B, fraction A) could be removed after storage at 20 C. We
hypothesized that as a result of the
freeze-thaw step, contaminating cells
lyse, but their nuclei remain intact
and can still be eliminated by highspeed centrifugation. Indeed, highspeed centrifugation of fraction A
plasma samples after storage completely eliminated the cellular contamination (Fig. 1B, fraction C). The
efficiency of elimination was identical to that of high-speed centrifugation before storage (Fig. 1B, fraction
B).
We conclude that the efficiency of
removal of contaminating cellular
DNA from plasma is dependent on
many preanalytical factors, including
centrifugal force, amount of collected
blood, and pipetting efficacy. Most
importantly, we have shown that irrespective of the protocol used for
plasma collection, contaminating

cells that remained in the plasma can

be removed after storage (20 C) of
samples. Therefore, frozen plasma
collections can be used retrospectively for DNA analysis when subjected to additional centrifugation at
16 000g after thawing.
References
1. Lo YMD. Fetal DNA in maternal plasma: biology
and diagnostic applications. Clin Chem 2000;
46:1903 6.
2. Anker P, Mulcahy H, Chen XQ, Stroun M. Detection of circulating tumour DNA in the blood
(plasma/serum) of cancer patients. Cancer Metastasis Rev 1999;18:6573.
3. Swinkels DW, de Kok JB, Hendriks JCM, Wiegerinck E, Zusterzeel PLM, Steegers EAP. Hemolysis, elevated liver enzymes, and low platelet
count (HELLP) syndrome as a complication of
preeclampsia in pregnant women increases the
amount of cell-free fetal and maternal DNA in
maternal plasma and serum. Clin Chem 2002;
48:650 3.
4. Sozzi G, Conte D, Mariani L, Lo Vullo S, Roz L,
Lombardo C, et al. Analysis of circulating tumor
DNA in plasma at diagnosis and during follow-up
of lung cancer patients. Cancer Res 2001;61:
4675 8.
5. Chiu RWK, Poon LLM, Lau TK, Leung TN, Wong
EMC, Lo YMD. Effects of blood-processing protocols on fetal and total DNA quantification in
maternal plasma. Clin Chem 2001;47:160713.

Dorine W. Swinkels1*
Erwin Wiegerinck1
Eric A.P. Steegers2
Jacques B. de Kok1
1
Department of Clinical Chemistry
University Medical Centre Nijmegen
6500 HB Nijmegen, The Netherlands
2

Department of Obstetrics
and Gynaecology
University Hospital Rotterdam
3000 CA Rotterdam, The Netherlands
*Address correspondence to this author at: Department of Clinical Chemistry/564, University Medical Centre
Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Fax 31-243541743;
e-mail D.Swinkels@akc.umcn.nl.