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25
| 1 | 2008
Klinisk Biokemi i Norden
(Fortsætter side 26)
and real-time PCR based assays for quantification of
the panels in clinical settings, may be valuable tools in
the future.
As for today qRT-PCR assays are most established
for detection of viral load and therapy monitoring
(HIV, SARS) (28-31) and for diagnosis and detection
of disease-specific prognostics markers in patients
with leukaemia (32;33).
Circulating extra-cellular DNA and RNA
General considerations
The discovery of cell-free nucleic acids in plasma was
first reported by Mandel and Metais in 1948 (34), but
was initially not widely recognized.
By the term circulating extra-cellular nucleic acids,
we imply DNA and RNA that exist in blood plasma
as free nucleic acid. Thus, extra-cellular nucleic acids
have escaped their natural environment and have
gained access to plasma where they may exist in solu-
tion or may be particle-bound. These forms of circu-
lating extracellular nucleic acids therefore should be
considered apart from circulating cellular nucleic acids
as regards biomarker functionality.
Methodological considerations
Since circulating extra-cellular nucleic acids may exist
in solution in plasma in various particular forms or
bound to blood cells, special analytical considerations
have to be taken into considerations in order to obtain
comparable results. As of today most studies have been
performed on plasma processed from EDTA whole
blood or serum from clotted blood (35). We have
found no studies that have systematically compared
the effects of various anticoagulants. A few compara-
tive studies using both plasma and serum have been
done (36), the results usually giving higher and more
labile serum than plasma values (37), most likely due
to the release of cellular constituents upon inclusion
of blood cells into the clot. For quantitative estimates
we would recommend the use of plasma obtained by
centrifugation (1600xg, 10 min, 4oC, plasma pipetted
off and recentrifuged 16000xg, 10 min 4oC (35;36))
of EDTA (5 mmol/L) whole blood. EDTA blood,
prior to processing may be stored at +4C for up to
24 hours prior to processing. Only a few studies on
extra-cellular circulating nucleic, cell-bound (38) or
filterable (39), have been reported. Since circulating
nucleic acid investigations frequently implies its use in
epidemiological studies where sample number often
may be quite large, automated extraction from plasma
has been advocated (35).
Circulating extra-cellular DNA and RNA in disease
General comments
At present there are two major fields where research –
on the brink of routine in some places – push on. The
first is focused on understanding fetal genetic make up
and wellbeing by examining for fetal DNA and RNA
in maternal plasma. The other is directed at early or
supplementary cancer diagnostics. Most studies in
these fields have extracted nucleic acids from plasma
(35;37)
Fetal DNA and RNA in maternal plasma
Conventional methods of obtaining fetal tissues for
genetic analysis, including amniocentesis and cho-
Foto: Henrik Alfthan. Island.
