U.S. patent application number 14/347051 was filed with the patent office on 2014-08-14 for stabilisation and isolation of extracellular nucleic acids.
This patent application is currently assigned to Qiagen GmbH. The applicant listed for this patent is Qiagen GmbH. Invention is credited to Martin Horlitz, Anabelle Schubert, Markus Sprenger-Haussels.
Application Number | 20140227688 14/347051 |
Document ID | / |
Family ID | 47994307 |
Filed Date | 2014-08-14 |
United States Patent
Application |
20140227688 |
Kind Code |
A1 |
Horlitz; Martin ; et
al. |
August 14, 2014 |
STABILISATION AND ISOLATION OF EXTRACELLULAR NUCLEIC ACIDS
Abstract
The present invention provides methods, compositions and devices
for stabilizing the extracellular nucleic acid population in a
cell-containing biological sample using an apoptosis inhibitor,
preferably a caspase inhibitor, a hypertonic agent and/or a
compound according to formula (1) as defined in the claims.
Inventors: |
Horlitz; Martin; (Hilden,
DE) ; Schubert; Anabelle; (Hilden, DE) ;
Sprenger-Haussels; Markus; (Hilden, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Qiagen GmbH |
Hilden |
|
DE |
|
|
Assignee: |
Qiagen GmbH
Hilden
DE
|
Family ID: |
47994307 |
Appl. No.: |
14/347051 |
Filed: |
September 25, 2012 |
PCT Filed: |
September 25, 2012 |
PCT NO: |
PCT/EP2012/068892 |
371 Date: |
March 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61539245 |
Sep 26, 2011 |
|
|
|
Current U.S.
Class: |
435/6.1 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12N 15/1003 20130101; C12Q 2527/125 20130101; C12Q 1/68 20130101;
C12Q 1/6806 20130101 |
Class at
Publication: |
435/6.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 26, 2011 |
EP |
11182819.0 |
Claims
1. A method for stabilizing an extracellular nucleic acid
population comprised in a cell-containing sample by contacting a
sample with a caspase inhibitor.
2. The method according to claim 1, comprising contacting the
sample with a) at least one caspase inhibitor; b) optionally at
least one hypertonic agent which stabilizes the cells comprised in
the sample; and c) optionally at least one compound according to
formula 1, ##STR00009## wherein R1 is a hydrogen residue or an
alkyl residue, preferably a C1-C5 alkyl residue, more preferred a
methyl residue, R2 and R3 are identical or different hydrocarbon
residues with a length of the carbon chain of 1-20 atoms arranged
in a linear or branched manner, and R4 is an oxygen, sulphur or
selenium residue.
3. The method according to claim 1 or 2, wherein the sample is
additionally contacted with an anticoagulant.
4. The method according to one or more of claims 1 to 3, wherein
the release of genomic DNA from cells contained in the sample into
the cell-free portion of the sample is reduced and/or the
degradation of nucleic acids present in the sample is reduced due
to the stabilization.
5. The method according to one or more of claims 1 to 4, wherein a)
the caspase inhibitor has one or more of the following
characteristics: i) it is a pancaspase inhibitor; and/or ii) it is
selected from the group consisting of Q-VD-OPh and
Z-Val-Ala-Asp(OMe)-FMK and/or b) the hypertonic agent has one or
more, preferably two or more of the following characteristics: i)
it is uncharged; ii) it stabilizes the cells comprised in the
sample by inducing cell shrinking; iii) it is cell impermeable; iv)
it is water-soluble; v) it is a hydroxylated organic compound; vi)
it is a polyol; vii) it is a hydroxy-carbonyl compound; viii) it is
a carbohydrate or a sugar alcohol; and/or ix) it is
dihydroxyacetone and/or c) wherein the compound according to
formula 1 has one or more of the following characteristics: i) R1,
R2 and R3 comprise 1 to 5 carbon atoms; ii) R1, R2 and R3 comprise
1 or 2 carbon atoms; iii) R4 is oxygen; iv) it is a
N,N-dialkyl-carboxylic acid amide; v) it is selected from the group
consisting of N,N-dimethylacetamide, N,N-diethylacetamide,
N,N-dimethylformamide and N,N-diethylformamide; and/or vi) it is
N,N-dimethylpropanamide.
6. The method according to one or more of claims 1 to 5, wherein
the sample is additionally contacted with at least one
anticoagulant, preferably a chelating agent such as EDTA.
7. The method according to one or more of claims 1 to 6, wherein
after the sample has been contacted with the caspase inhibitor, the
hypertonic agent, the compound according to formula 1 and/or the
anticoagulant, the resulting mixture has one or more of the
following characteristics: a) it comprises the caspase inhibitor in
a concentration selected from at least 0.01 .mu.M, at least 0.05
.mu.M, at least 0.1 .mu.M, at least 0.5 .mu.M, at least 1 .mu.M, at
least 2.5 .mu.M or at least 3.5 .mu.M; b) it comprises the caspase
inhibitor in a concentration range selected from 0.01 .mu.M to 100
.mu.M, 0.05 .mu.M to 100 .mu.M, 0.1 .mu.M to 50 .mu.M, 1 .mu.M to
40 .mu.M, 1 .mu.M to 30 .mu.M or 2.5 .mu.M to 25 .mu.M; c) it
comprises the hypertonic agent in a concentration of at least
0.05M, at least 0.1 M, preferably at least 0.25M, more preferably
at least 0.5M; d) it comprises the hypertonic agent in a
concentration range selected from 0.05M to 2M, 0.1 to 1.5M, 0.15M
to 0.8M, 0.2M to 0.7M or 0.1M to 0.6M; e) it comprises the compound
according to formula 1 in a concentration of at least 0.1%, at
least 0.5%, at least 1%, at least 0.75%, at least 1%, at least
1.25% or at least 1.5%; f) it comprises the compound according to
formula 1 in a concentration range selected from 0.1% to 50%, 0.5%
to 25%, 0.75% to 20%, 1% to 15% or 1% to 10%; and/or g) it
comprises the anticoagulant, preferably a chelating agent, in a
concentration range selected from 0.05 mM to 100 mM, 0.05 mM to 50
mM, 0.1 mM to 30 mM, 1 mM to 20 mM or 2 mM to 15 mM.
8. The method according to one or more of claims 1 to 7, wherein
the sample is for stabilization contacted with: a) at least one
pancaspase inhibitor as caspase inhibitor, b) at least one
hypertonic agent, preferably a hydroxylated organic compound and c)
optionally at least one compound according to formula 1, preferably
an N,N-dialkyl-carboxylic acid amide, and d) optionally an
anticoagulant, preferably a chelating agent, more preferably EDTA,
wherein the compounds according to a) to d) are comprised in a
stabilising composition.
9. The method according to one or more of claims 1 to 8, wherein
the sample has one or more of the following characteristics: a) it
comprises extracellular nucleic acids; b) it is selected from the
group consisting of whole blood, samples derived from blood,
plasma, serum, lymphatic fluid, urine, liquor, cerebrospinal fluid,
ascites, milk, stool, bronchial lavage, saliva, amniotic fluid,
semen/seminal fluid, swabs/smears, body fluids, body secretions,
nasal secretions, vaginal secretions, wound secretions and
excretions and cell culture supernatants; c) it is a cell-depleted
or cell-containing body fluid; d) it is selected from whole blood,
plasma and/or serum; and/or e) it is whole blood.
10. The method according to one or more of claims 1 to 9, wherein
stabilization of the extracellular nucleic acid population is
achievable without refrigeration, preferably at room temperature,
for a time period selected from a) at least two days; b) at least
three days; c) at least one day to three days; d) at least one day
to six days; and/or e) at least one day to seven days.
11. The method according to one or more of claims 1 to 10, wherein
a) the one or more stabilising agents and optionally further
additives are comprised in a stabilising composition and wherein
the volumetric ratio of the stabilising composition to the
specified volume of the cell-containing sample is selected from
10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5; b) the
stabilized sample is subjected to a nucleic acid analysis and/or
detection method; c) extracellular nucleic acids are isolated from
the stabilized sample; d) extracellular nucleic acids are isolated
from the stabilized sample and the isolated nucleic acids are
analysed and/or detected; e) cells comprised in the stabilized
sample are removed; f) cells comprised in the stabilized sample are
removed prior to performing an isolation, analysis and/or detection
step; g) a nucleic acid isolation step is performed after a
stabilization period as defined in claim 10; h) (i) the stabilized
sample, (ii) the stabilized sample from which cells have been
removed and/or (iii) cells removed from the sample are stored; i)
cells that were removed from the stabilized sample are discarded;
and/or j) nucleic acids are isolated from cells that were removed
from the stabilized sample.
12. The method according to one or more of claims 1 to 11, for
stabilizing an extracellular nucleic acid population comprised in a
blood sample, comprising contacting the blood sample with a caspase
inhibitor and an anticoagulant, wherein the release of genomic DNA
from cells contained in the blood sample into the cell-free portion
of the blood sample is reduced and the degradation of nucleic acids
present in the sample is reduced due to the stabilization.
13. A method for isolating extracellular nucleic acids from a
biological sample, preferably a blood sample, comprising the steps
of: a) stabilizing the extracellular nucleic acid population in the
sample according to the method defined in one or more of claims 1
to 12; and b) isolating extracellular nucleic acids.
14. The method according to claim 13, comprising one or more of the
following steps: i) optionally removing cells from the
cell-containing sample between step a) and step b); ii) performing
one or more of the steps b) to j) as defined in claim 11; and/or
iii) isolating extracellular nucleic acids from the sample in step
b) of claim 13 using an isolation method selected from the group
comprising extraction, solid-phase extraction, isolation methods
using a nucleic acid binding solid phase, isolation methods using a
silica material, isolation methods that are based on the use of a
solid phase comprising anionic exchange groups; magnetic
particle-based purification, phenol-chloroform extraction, alcohol
and/or chaotropic agent(s) based nucleic isolation method,
chromatography, anion-exchange chromatography, anion exchange
particle-based isolation, electrophoresis, filtration,
precipitation, target nucleic acid specific isolation methods and
combinations thereof.
15. The method according to claim 13 or 14, wherein the isolated
nucleic acids are in a further step c) processed and/or analysed
and preferably are: i) modified; ii) contacted with at least one
enzyme; iii) amplified; iv) reverse transcribed; v) cloned; vi)
sequenced; vii) contacted with a probe; viii) detected; ix)
quantified; and/or ix) identified.
16. The method according to one or more of claims 13 to 15, wherein
a) the extracellular nucleic acid population that is isolated from
the cell-free portion of the sample and/or that is obtained after
isolation in step b) of claim 13, has one or more of the following
characteristics: i) it is comprised as a portion in the total
nucleic acid that is isolated; ii) it predominantly comprises DNA;
iii) it predominantly comprises RNA; iv) it comprises circulating
extracellular nucleic acids; v) it comprises disease related
nucleic acids; vi) it comprises tumor-associated or tumor-derived
nucleic acids; vii) it comprises inflammation related nucleic
acids: viii) it comprises fetal nucleic acids; ix) it comprises
viral nucleic acids; x) it comprises pathogen nucleic acids; xi) it
comprises mammalian extracellular nucleic acids; and/or xii) it is
a mixture of DNA and RNA; and/or b) the extracellular nucleic acid
that is analysed and/or further processed, preferably detected, in
step c), has one or more of the following characteristics: i) it is
DNA; ii) it is RNA; iii) it is a circulating extracellular nucleic
acid; iv) it comprises disease related nucleic acids; v) it
comprises tumor-associated or tumor-derived nucleic acids; vi) it
comprises inflammation related nucleic acids: vii) it is a fetal
nucleic acid; viii) it is a viral nucleic acid; ix) it is a
pathogen nucleic acid; x) it is a mammalian extracellular nucleic
acid; and/or xi) it is a mixture of DNA and RNA;
17. A composition suitable for stabilizing the extracellular
nucleic acid population in a biological sample, preferably a blood
sample, comprising a) at least one caspase inhibitor, and
comprising at least one further compound selected from b), c) and
d), wherein b) is at least one hypertonic agent suitable for
stabilizing cells comprised in the sample, preferably is at least
one hydroxylated organic compound; c) is at least one compound
according to formula 1, ##STR00010## wherein R1 is a hydrogen
residue or an alkyl residue, preferably a C1-C5 alkyl residue, more
preferred a methyl residue, R2 and R3 are identical or different
hydrocarbon residues with a length of the carbon chain of 1-20
atoms arranged in a linear or branched manner, and R4 is an oxygen,
sulphur or selenium residue; d) is at least one anticoagulant,
preferably a chelating agent.
18. The composition according to claim 17, comprising at least one
caspase inhibitor and at least one anticoagulant.
19. The composition according to claim 17 or 18, having one or more
of the following characteristics: a) it is capable of reducing the
release of genomic DNA from cells contained in the sample into the
cell-free portion of the sample; b) it is capable of reducing the
degradation of nucleic acids, in particular genomic DNA, present in
the sample; c) the caspase inhibitor has one or more of the
characteristics as defined in claim 5; d) it comprises at least one
hypertonic agent as defined in claim 5; e) it comprises at least
one compound according to formula 1 as defined in claim 5; f) when
mixed with a biological sample, preferably blood, plasma or serum,
the resulting mixture comprises the caspase inhibitor, the
hypertonic agent, the compound according to formula 1 and/or the
chelating agent in a concentration as defined in claim 7; g) it is
provided in a solid form; h) it is provided in a liquid form;
and/or i) it is capable of stabilizing the extracellular nucleic
acid population contained in said sample at room temperature for at
least 3 days, preferably at least 6 days.
20. The composition according to one or more of claims 17 to 19,
wherein the stabilizing composition is provided as mixture with a
biological sample and wherein said sample has one or more of the
following characteristics: a) it comprises extracellular nucleic
acids; b) it is selected from the group consisting of whole blood,
plasma, serum, lymphatic fluid, urine, liquor, cerebrospinal fluid,
ascites, milk, stool, bronchial lavage, saliva, amniotic fluid,
semen/seminal fluid, swabs/smears, body fluids, body secretions,
nasal secretions, vaginal secretions, wound secretions and
excretions and cell culture supernatants; c) it is a cell-depleted
or cell containing body fluid; d) it is selected from whole blood,
plasma and/or serum; and/or e) it is whole blood.
21. The composition according to claim 20, wherein volumetric ratio
of the stabilising composition to the specified volume of the
cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15,
1:1 to 1:10 and 1:2 to 1:5.
Description
[0001] The work leading to this invention has received funding from
the European Community's Seventh Framework Programme
(FP7/2007-2013) under grant agreement n.degree. 222916.
FIELD OF THE INVENTION
[0002] The technology disclosed herein relates to methods and
compositions suitable for stabilizing the extracellular nucleic
acid population in a cell-containing sample, in particular a blood
sample, and to a method for isolating extracellular nucleic acids
from respectively stabilized biological samples.
BACKGROUND
[0003] Extracellular nucleic acids have been identified in blood,
plasma, serum and other body fluids. Extracellular nucleic acids
that are found in respective samples are to a certain extent
degradation resistant due to the fact that they are protected from
nucleases (e.g. because they are secreted in form of a proteolipid
complex, are associated with proteins or are contained in
vesicles). The presence of elevated levels of extracellular nucleic
acids such as DNA and/or RNA in many medical conditions,
malignancies, and infectious processes is of interest inter alia
for screening, diagnosis, prognosis, surveillance for disease
progression, for identifying potential therapeutic targets, and for
monitoring treatment response. Additionally, elevated fetal DNA/RNA
in maternal blood is being used to determine e.g. gender identity,
assess chromosomal abnormalities, and monitor pregnancy-associated
complications. Thus, extracellular nucleic acids are in particular
useful in non-invasive diagnosis and prognosis and can be used e.g.
as diagnostic markers in many fields of application, such as
non-invasive prenatal genetic testing, oncology, transplantation
medicine or many other diseases and, hence, are of diagnostic
relevance (e.g. fetal- or tumor-derived nucleic acids). However,
extracellular nucleic acids are also found in healthy human beings.
Common applications and analysis methods of extracellular nucleic
acids are e.g. described in WO97/035589, WO97/34015, Swarup et al,
FEBS Letters 581 (2007) 795-799, Fleischhacker Ann. N.Y. Acad. Sci.
1075: 40-49 (2006), Fleischhacker and Schmidt, Biochmica et
Biophysica Acta 1775 (2007) 191-232, Hromadnikova et al (2006) DNA
and Cell biology, Volume 25, Number 11 pp 635-640; Fan et al (2010)
Clinical Chemistry 56:8.
[0004] Traditionally, the first step of isolating extracellular
nucleic acids from a cell-containing biological sample such as
blood is to obtain an essentially cell-free fraction of said
sample, e.g. either serum or plasma in the case of blood. The
extracellular nucleic acids are then isolated from said cell-free
fraction, commonly plasma, when processing a blood sample. However,
obtaining an essentially cell-free fraction of a sample can be
problematic and the separation is frequently a tedious and time
consuming multi-step process as it is important to use carefully
controlled conditions to prevent cell breakage during
centrifugation which could contaminate the extracellular nucleic
acids with cellular nucleic acids released during breakage.
Furthermore, it is often difficult to remove all cells. Thus, many
processed samples that are often and commonly classified as
"cell-free" such as plasma or serum in fact still contain residual
amounts of cells that were not removed during the separation
process. Another important consideration is that cellular nucleic
acid are released from the cells contained in the sample due to
cell breakage during ex vivo incubation, typically within a
relatively short period of time from a blood draw event. Once cell
lysis begins, the lysed cells release additional nucleic acids
which become mixed with the extracellular nucleic acids and it
becomes increasingly difficult to recover the extracellular nucleic
acids for testing. These problems are discussed in the prior art
(see e.g. Chiu et al (2001), Clinical Chemistry 47:9 1607-1613; Fan
et al (2010) and US2010/0184069). Further, the amount and
recoverability of available extracellular nucleic acids can
decrease substantially over a period of time due to
degradation.
[0005] Besides mammalian extracellular nucleic acids that derive
e.g. from tumor cells or the fetus, cell-containing samples may
also comprise other nucleic acids of interest that are not
comprised in cells. An important, non-limiting example is pathogen
nucleic acids such as viral nucleic acids. Preservation of the
integrity of viral nucleic acids in cell-containing samples such as
in particular in blood specimens during shipping and handling is
also crucial for the subsequent analysis and viral load
monitoring.
[0006] The above discussed problems particularly are an issue, if
the sample comprises a high amount of cells as is the case e.g.
with whole blood samples. Thus, in order to avoid respectively
reduce the above described problems it is common to separate an
essentially cell-free fraction of the sample from the cells
contained in the sample basically immediately after the sample is
obtained. E.g. it is recommended to obtain blood plasma from whole
blood basically directly after the blood is drawn and/or to cool
the whole blood and/or the obtained plasma or serum in order to
preserve the integrity of the extracellular nucleic acids and to
avoid contaminations of the extracellular nucleic acid population
with intracellular nucleic acids that are released from the
contained cells. However, the need to directly separate e.g. the
plasma from the blood is a major disadvantage because many
facilities wherein the blood is drawn (e.g. a doctor's practice) do
not have a centrifuge that would enable the efficient separation of
blood plasma. Furthermore, plasma that is obtained under regular
conditions often comprises residual amounts of cells which
accordingly, may also become damaged or may die during handling of
the sample, thereby releasing intracellular nucleic acids, in
particular genomic DNA, as is described above. These remaining
cells also pose a risk that they become damaged during the handling
so that their nucleic acid content, particularly genomic (nuclear)
DNA and cytoplasmic RNA, would merge with and thereby contaminate
respectively dilute the extracellular, circulating nucleic acid
fraction. To remove these remaining contaminating cells and to
avoid/reduce the aforementioned problems, it was known to perform a
second centrifugation step at higher speed. However, again, such
powerful centrifuges are often not available at the facilities
wherein the blood is obtained. Furthermore, even if plasma is
obtained directly after the blood is drawn, it is recommended to
freeze it at -80.degree. C. in order to preserve the nucleic acids
contained therein if the nucleic acids can not be directly
isolated. This too imposes practical constraints upon the
processing of the samples as e.g. the plasma samples must be
shipped frozen. This increases the costs and furthermore, poses a
risk that the sample gets compromised in case the cold chain is
interrupted.
[0007] Blood samples are presently usually collected in blood
collection tubes containing spray-dried or liquid EDTA (e.g. BD
Vacutainer K.sub.2EDTA). EDTA chelates magnesium, calcium and other
bivalent metal ions, thereby inhibiting enzymatic reactions, such
as e.g. blood clotting or DNA degradation due to DNases. However,
even though EDTA is an efficient anticoagulant, EDTA does not
efficiently prevent the dilution respectively contamination of the
extracellular nucleic acid population by released intracellular
nucleic acids. Thus, the extracellular nucleic acid population that
is found in the cell-free portion of the sample changes during the
storage. Accordingly, EDTA is not capable of sufficiently
stabilising the extracellular nucleic acid population in particular
because it can not avoid the contamination of the extracellular
nucleic acid population with e.g. genomic DNA fragments which are
generated after blood draw by cell degradation and cell instability
during sample transportation and storage.
[0008] Methods are known in the prior art that specifically aim at
stabilizing circulating nucleic acids contained in whole blood. One
method employs the use of formaldehyde to stabilize the cell
membranes, thereby reducing the cell lysis and furthermore,
formaldehyde inhibits nucleases. Respective methods are e.g.
described in U.S. Pat. No. 7,332,277 and U.S. Pat. No. 7,442,506.
However, the use of formaldehyde or formaldehyde-releasing
substances has drawbacks, as they may compromise the efficacy of
extracellular nucleic acid isolation by induction of crosslinks
between nucleic acid molecules or between proteins and nucleic
acids. Alternative methods to stabilize blood samples are described
e.g. in US 2010/0184069 and US 2010/0209930. These rather recently
developed methods demonstrate the great need for providing means to
stabilise cell-containing biological samples, to allow the
efficient recovery of e.g. extracellular nucleic acids contained in
such samples.
[0009] However, despite these rather recent developments there is
still a continuous need to develop sample processing techniques
which result in a stabilisation of the extracellular nucleic acid
population comprised in a biological sample, in particular a sample
containing cells, including samples suspected of containing cells,
in particular whole blood, plasma or serum, thereby making the
handling, respectively processing of such samples easier (e.g. by
avoiding the need to directly separate plasma from whole blood or
to cool or even freeze the isolated plasma) thereby also making the
isolation and testing of extracellular nucleic acids contained in
such samples more reliable and consequently, thereby improving the
diagnostic and prognostic capabilities of the extracellular nucleic
acids. In particular, there is a continuous need for a solution for
preserving extracellular nucleic acids in whole blood samples, e.g.
for prenatal testing and/or for screening for neoplastic, in
particular premalignant or malignant diseases.
[0010] It is the object of the present invention to overcome at
least one of the drawbacks of the prior art sample stabilization
methods. Thus, it is inter alia an object of the present invention
to provide a method that is capable of stabilising a
cell-containing sample, in particular whole blood. In particular,
it is an object of the present invention to stabilise the
extracellular nucleic acid population contained in a biological
sample and in particular to avoid a contamination of the
extracellular nucleic acid population with genomic DNA, in
particular fragmented genomic DNA. Furthermore, it is in particular
an object of the present invention to provide a method suitable for
stabilising a biological sample, preferably a whole blood sample,
even at room temperature, preferably for a period of at least two,
preferably at least three days. Furthermore, it is an object of the
present invention to provide a sample collection container, in
particular a blood collection tube that is capable of effectively
stabilising a biological sample and in particular the extracellular
nucleic acid population comprised in the sample.
SUMMARY OF THE INVENTION
[0011] The present invention is based on the finding that certain
additives are surprisingly effective in stabilizing cell-containing
biological samples comprising extracellular nucleic acids, in
particular whole blood samples or samples derived from whole blood
such as e.g. blood plasma. It was found that these additives are
highly efficient in stabilizing the extracellular nucleic acid
population and in particular are capable to avoid or at least
significantly reduce contaminations with genomic DNA, in particular
fragmented genomic DNA.
[0012] According to a first aspect, a method suitable for
stabilizing an extracellular nucleic acid population comprised in a
cell-containing sample is provided, wherein a sample is contacted
with [0013] a) at least one apoptosis inhibitor, [0014] b) at least
one hypertonic agent, which stabilizes the cells comprised in the
sample, and/or [0015] c) at least one compound according to formula
1
##STR00001##
[0015] wherein R1 is a hydrogen residue or an alkyl residue,
preferably a C1-C5 alkyl residue, more preferred a methyl residue,
R2 and R3 are identical or different hydrocarbon residues with a
length of the carbon chain of 1-20 atoms arranged in a linear or
branched manner, and R4 is an oxygen, sulphur or selenium
residue.
[0016] According to a first sub-aspect, a method suitable for
stabilizing an extracellular nucleic acid population comprised in a
cell-containing sample is provided, wherein the sample is contacted
with at least one apoptosis inhibitor. Preferably, the
cell-containing sample is selected from whole blood, plasma or
serum. Surprisingly, it was found that the apoptosis inhibitor
reduces contaminations of the extracellular nucleic acid population
with intracellular nucleic acids, in particular fragmented genomic
DNA, that originate from cells contained in the sample, e.g. from
damaged or dying cells. Furthermore, the inventors found that the
apoptosis inhibitor reduces the degradation of nucleic acids
present in the sample. Thus, the stabilization according to the
present invention using an apoptosis inhibitor has the effect that
the extracellular nucleic acid population contained in the sample
is substantially preserved in the state it had shown at the time
the biological sample was obtained, respectively collected.
[0017] According to a second sub-aspect, a method suitable for
stabilizing an extracellular nucleic acid population comprised in a
cell-containing sample is provided, wherein a sample is contacted
with at least one hypertonic agent, which is capable of stabilizing
cells comprised in the sample. It was surprisingly found that cell
shrinking that is induced by mild hypertonic effects (osmosis)
results in a considerable increase of the cell stability. By
increasing the cell stability, the hypertonic agent in particular
reduces the release of intracellular nucleic acids, in particular
genomic DNA, from the contained cells into the extracellular
portion or compartment of the sample. Thus, the stabilization
according to the present invention using a hypertonic agent has the
effect that the extracellular nucleic acid population contained in
the sample is substantially preserved in the state it had shown at
the time the biological sample was obtained, respectively
collected.
[0018] According to a third sub-aspect of the present invention, a
method suitable for stabilizing an extracellular nucleic acid
population comprised in a cell-containing sample is provided,
wherein a sample is contacted with at least one compound according
to formula 1
##STR00002##
wherein R1 is a hydrogen residue or an alkyl residue, preferably a
C1-C5 alkyl residue, more preferred a methyl residue, R2 and R3 are
identical or different hydrocarbon residues with a length of the
carbon chain of 1-20 atoms arranged in a linear or branched manner,
and R4 is an oxygen, sulphur or selenium residue. It was found that
adding a respective compound as an advantageous stabilizing effect
on the extracellular nucleic acid population.
[0019] According to a fourth sub-aspect, a method suitable for
stabilizing an extracellular nucleic acid population comprised in a
cell-containing sample is provided, wherein a sample is contacted
with [0020] a) at least one apoptosis inhibitor, and [0021] b) at
least one hypertonic agent, which stabilizes the cells comprised in
the sample.
[0022] It was found that the combination of these stabilizing
agents (and optionally further additives) is remarkably effective
in inhibiting the release of intracellular nucleic acids, in
particular genomic DNA, from the contained cells into the
extracellular portion of the sample. Furthermore, it was shown that
the degradation of nucleic acids present in the sample is highly
efficiently prevented. In particular, less fragmented genomic DNA
is found in respectively stabilized samples. Thus, the
stabilization according to the present invention using this
combination of stabilizing additives has the effect that the
extracellular nucleic acid population contained in the sample is
substantially and effectively preserved in the state it had shown
at the time the biological sample was obtained, respectively
collected (e.g. drawn in the case of blood) and that in particular
contaminations of the extracellular nucleic acid population with
fragmented genomic DNA are reduced.
[0023] In order to enhance the stabilization effect towards
extracellular nucleic acids, it is also an object of the present
invention to provide further combinations of stabilizing agents in
order to stabilize the extracellular nucleic acid population
comprised in a cell-containing sample. A respective combination may
comprise at least one apoptosis inhibitor, at least one hypertonic
agent and/or at least one compound according to formula 1 as
defined above, for example (1) a combination of at least one
apoptosis inhibitor and at least one compound according to formula
1 as defined above, (2) a combination of at least one hypertonic
agent and at least one compound according to formula 1 or (3) a
combination of all three stabilizing agents, i.e. at least one
apoptosis inhibitor, at least one hypertonic agent and at least one
compound according to formula 1. A respective combination may also
comprise additional additives that enhance the stabilizing effect
such as e.g. chelating agents. In case the sample is blood or a
sample derived from blood, usually an anticoagulant is also added.
Chelating agents such as e.g. EDTA are suitable for this purpose.
Respective stabilizing combinations can be according to a fifth
sub-aspect advantageously used in a method suitable for stabilizing
an extracellular nucleic acid population comprised in a
cell-containing sample according to the first aspect of the present
invention.
[0024] According to a second aspect, a method for isolating
extracellular nucleic acids from a biological sample is provided,
wherein said method comprises the steps of: [0025] a) stabilizing
the extracellular nucleic acid population comprised in a sample
according to the method defined in the first aspect of the present
invention; and [0026] b) isolating extracellular nucleic acids from
said sample.
[0027] Stabilization in step a) can be achieved e.g. according to
one of the five sub-aspects of the first aspect according to the
present invention as described above. As discussed above, the
stabilization according to the present invention has the effect
that the extracellular nucleic acid population contained in the
sample is substantially preserved in the state it had shown at the
time the biological sample was obtained, respectively collected.
Therefore, extracellular nucleic acids obtained from a respectively
stabilized sample comprise less contaminations with intracellular
nucleic acids, in particular fragmented genomic DNA, that results
e.g. from decaying cells comprised in the sample compared to
extracellular nucleic acids that are obtained from an unstabilized
sample. The substantial preservation of the extracellular nucleic
acid population is an important advantage because this
stabilization/preservation enhances the accuracy of any subsequent
tests. It allows for standardizing the isolation and subsequent
analysis of the extracellular nucleic acid population, thereby
making diagnostic or prognostic applications that are based on the
extracellular nucleic acid fraction more reliable and more
independent from the used storage/handling conditions. Thereby, the
diagnostic and prognostic applicability of the respectively
isolated extracellular nucleic acids is improved. In particular,
the teachings of the present invention have the advantage that the
ratio of certain extracellular nucleic acid molecules can be kept
substantially constant compared to the ratio at the time the sample
was collected. The stabilization achieves that intracellular
nucleic acids are substantially kept within the cells and that
extracellular nucleic acids are substantially stabilized.
[0028] According to a third aspect, a composition suitable for
stabilizing a cell-containing biological sample is provided,
comprising: [0029] a) at least one apoptosis inhibitor, preferably
a caspase inhibitor, and/or [0030] b) at least one hypertonic agent
which is suitable for stabilizing the cells comprised in the
sample, preferably a hydroxylated organic compound; and/or [0031]
c) at least one compound according to formula 1 as defined above;
and/or [0032] d) optionally at least one anticoagulant, preferably
a chelating agent.
[0033] A respective stabilizing composition is particularly
effective in stabilizing a cell-containing biological sample, in
particular whole blood, plasma and/or serum by stabilizing the
cells and the extracellular nucleic acid population comprised in
said sample. Preferably, at least two of the stabilizing agents
defined in a) to c) more preferred all of the stabilizing agents
defined in a) to c) are present in the stabilizing composition. A
respective stabilizing composition allows the storage and/or
handling, e.g. shipping, of the sample, e.g. whole blood, at room
temperature for at least two, or preferably at least three days
without substantially compromising the quality of the sample,
respectively the extracellular nucleic acid population contained
therein. Thus, when using the stabilization composition according
to the present invention, the time between sample collection, e.g.
blood collection, and nucleic acid extraction can vary without
substantial effect on the extracellular nucleic acid population
contained in the sample. This is an important advantage as it
reduces the variability in the extracellular nucleic acid
population attributable to different handling procedures.
[0034] According to a forth aspect, a container for collecting a
cell-containing biological sample, preferably a blood sample, is
provided wherein the container comprises a composition according to
the third aspect of the present invention. Providing a respective
container, e.g. a sample collection tube comprising the stabilizing
composition has the advantage that the sample is immediately
stabilized as soon as the sample is collected in the respective
container. Furthermore, a respective sample collection container,
in particular a blood collection tube, is capable of stabilising
blood cells and extracellular nucleic acids and optionally, viruses
respectively viral nucleic acids contained in a blood sample or a
sample derived from blood. Thereby, a further problem was
overcome.
[0035] According to a fifth aspect, a method is provided comprising
the step of collecting, preferably withdrawing, a biological
sample, preferably blood, from a patient directly into a chamber of
a container according to the fourth aspect of the present
invention.
[0036] According to a sixth aspect, a method of producing a
composition according to the third aspect of the present invention
is provided, wherein the components of the composition are mixed,
preferably are mixed in a solution. The term "solution" as used
herein in particular refers to a liquid composition, preferably an
aqueous composition. It may be a homogenous mixture of only one
phase but it is also within the scope of the present invention that
a solution comprises solid components such as e.g.
precipitates.
[0037] Other objects, features, advantages and aspects of the
present application will become apparent to those skilled in the
art from the following description and appended claims. It should
be understood, however, that the following description, appended
claims, and specific examples, while indicating preferred
embodiments of the application, are given by way of illustration
only. Various changes and modifications within the spirit and scope
of the disclosed invention will become readily apparent to those
skilled in the art from reading the following.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1a shows a gel picture after chip electrophoresis of
DNA isolated from samples treated with caspase inhibitors (Example
1).
[0039] FIG. 1b is a diagram showing the effect of caspase
inhibitors on the increase of ribosomal 18S DNA in plasma (Example
1).
[0040] FIG. 2a shows a gel picture after chip electrophoresis of
DNA isolated from samples treated with different concentrations of
the caspase inhibitor Q-VD-OPH in combination (Example 2).
[0041] FIG. 2b is a diagram showing the effects of different
concentrations of the caspase-inhibitor Q-VD-OPH in combination
with glucose on the increase of ribosomal 18S DNA in the plasma
(Example 2).
[0042] FIG. 3 shows the blood cell integrity measured by flow
cytometry for blood cells treated with dihydroxyacetone dissolved
in different buffers (Example 3).
[0043] FIG. 4a shows a gel picture after chip electrophoresis of
DNA isolated from samples treated with dihydroxyacetone dissolved
in different buffers (Example 3).
[0044] FIG. 4b is a diagram showing the effect of dihydroxyacetone
on the increase of ribosomal 18S DNA (Example 3).
[0045] FIG. 5 shows the blood cell integrity measured by flow
cytometry for blood cells treated with different concentrations of
dihydroxyacetone (Example 4).
[0046] FIG. 6a shows a gel picture after chip electrophoresis of
DNA isolated from samples treated with different concentrations of
dihydroxyacetone (Example 4).
[0047] FIG. 6b is a diagram showing the effect of different
dihydroxyacetone concentrations on the increase of ribosomal 18S
DNA (Example 4).
[0048] FIG. 7a shows the blood cell integrity measured by flow
cytometry for blood cells treated with a combination of elevated
K.sub.2EDTA, Q-VD-OPH and DHA (Example 5).
[0049] FIG. 7b is a diagram showing the effect of the combination
of EDTA, DHA and Q-VD-OPH on the increase of 18S DNA (Example
5).
[0050] FIG. 8 is a diagram showing the effect of the combination of
EDTA, DHA and Q-VD-OPH on the transcript level of free circulating
mRNA in plasma (Example 6).
[0051] FIG. 9 is a diagram showing the effects of different
concentrations of DMAA on the increase of ribosomal 18S DNA in the
plasma.
[0052] FIG. 10 is a diagram showing the influence of different
sugar alcohols on the increase of 18S rDNA (Example 8)
[0053] FIG. 11 is a diagram showing the influence of substances on
the increase of 18S rDNA (Example 9)
[0054] FIG. 12 is a diagram showing the influence of substances on
the increase of 18S rDNA (Example 10)
[0055] FIG. 13 is a diagram showing the influence of substances on
the increase of 18S rDNA (Example 11)
[0056] FIG. 14 is a diagram showing the influence of substances on
the increase of 18S rDNA (Example 11)
[0057] FIG. 15 is a diagram showing the influence of substances on
the increase of 18S rDNA
[0058] FIG. 16 is a diagram showing the ccfDNA increase in plasma
fraction of whole blood incubated for up to 6 days at 37.degree. C.
(Example 13)
[0059] FIG. 17 is a diagram showing the ccfDNA increase in plasma
fraction of whole blood incubated for up to 6 days at 37.degree. C.
(Example 13)
[0060] FIG. 18 is a diagram showing the percent hits of spiked-in
DNA fragments (Example 14)
[0061] FIG. 19 is a diagram showing the mean copies (Example
14)
[0062] FIG. 20 is a diagram showing the percent of 18S compared to
BD Vacutainer K2E (Example 14)
[0063] FIG. 21 is a diagram showing the decrease of HIV, incubated
in whole blood at 37.degree. C., purified from plasma (Example
15)
[0064] FIG. 22 is a diagram showing the decrease of HCV, incubated
in whole blood at 37.degree. C., purified from plasma (Example
15)
[0065] FIG. 23 is a diagram showing the influence of propionamid on
18S rDNA increase Donor 1 (Example 16)
[0066] FIG. 24 is a diagram showing the influence of propionamid on
18S rDNA increase Donor 2 (Example 16)
DETAILED DESCRIPTION OF THIS INVENTION
[0067] The present invention is directed to methods, compositions
and devices and thus to technologies suitable for stabilizing the
extracellular nucleic acid population comprised in a
cell-containing biological sample. The stabilization technologies
disclosed herein reduce the risk that the extracellular nucleic
acid population is contaminated with intracellular nucleic acids,
in particular fragmented genomic DNA, which derives from, e.g. is
released from damaged and/or dying cells contained in the sample.
Therefore, the present invention achieves the stabilization of the
sample and hence the stabilization of the extracellular nucleic
acid population comprised therein without the lysis of the
contained cells. Rather, cells contained in the sample are
stabilized thereby substantially preventing or reducing the release
of intracellular nucleic acids. The remarkable stabilization that
is achieved with the methods and compositions of the present
invention allows the storage and/or handling of the stabilized
sample for a prolonged period of time at room temperature without
jeopardizing the quality of the sample, respectively the
extracellular nucleic acids contained therein. As the composition
of the extracellular nucleic acid population is stabilized and thus
substantially preserved at the time the sample is obtained by using
the teachings of the present invention, the time between sample
collection and nucleic acid extraction can vary without significant
effect on the composition of the extracellular nucleic acids
population. This allows the standardization of e.g. diagnostic or
prognostic extracellular nucleic acid analysis because variations
in the handling/storage of the samples have less influence on the
quality, respectively the composition of the extracellular nucleic
acid population, thereby providing an important advantage over
prior art methods. Hence, the samples, respectively the
extracellular nucleic acids obtained from respectively stabilized
samples become more comparable. Furthermore, the teachings of the
present invention obviate the necessity to directly separate cells
contained in the sample from the cell-free portion of the sample in
order to avoid, respectively reduce contaminations of the
extracellular nucleic acids with intracellular nucleic acids, in
particular fragmented genomic DNA, that is otherwise released from
decaying cells. This advantage considerably simplifies the handling
of the samples, in particular the handling of whole blood samples.
E.g. whole blood samples obtained in a clinic and stabilized
according to the teachings of the present invention can be shipped
at room temperature and the plasma containing the extracellular
nucleic acids can be conveniently separated in the receiving
clinical lab. However, the teachings of the invention are also
advantageous when processing cell-depleted biological samples, or
samples commonly referred to as "cell-free" such as e.g. blood
plasma or serum. Respective cell-depleted or "cell-free" biological
samples may still (also depending on the used separation process)
comprise residual cells, in particular white blood cells which
comprise genomic DNA, which accordingly, pose a risk that the
extracellular nucleic acid population becomes increasingly
contaminated with intracellular nucleic acids, in particular
fragmented genomic DNA, if the (potentially) remaining cells are
damaged or die during the shipping of storing process. This risk is
considerably reduced when using the stabilization method taught by
the present invention. Because the technology of the present
invention allows to efficiently preserve the extracellular nucleic
acid population of the sample at the time the sample is collected
and contacted with the stabilizing agents, said samples can be
properly worked up in the receiving facilities in order to isolate
the extracellular nucleic acids from said samples while
substantially avoiding respectively reducing contaminations of the
extracellular nucleic population with intracellular nucleic acids.
The facilities receiving the samples such as e.g. laboratories
usually also have the necessary equipment such as e.g. high speed
centrifuges (or other means, see also below) to efficiently remove
cells comprised in the samples, including residual cells that might
be present in cell-depleted samples such as e.g. in blood plasma.
Such equipment is often not present in the facilities where the
sample is obtained. Thus, the present invention has many advantages
when stabilizing biological samples which comprise a large amount
of cells such as e.g. whole blood samples, but also has important
advantages when stabilizing biological samples which comprise only
a small amount of cells or which may only be suspected of
containing cells such as e.g. plasma, serum, urine, saliva,
synovial fluids, amniotic fluid, lachrymal fluid, ichors, lymphatic
fluid, liquor, cerebrospinal fluid and the like.
[0068] According to a first aspect, a method suitable for
stabilizing the extracellular nucleic acid population comprised in
a cell-containing sample, preferably a blood sample, is provided,
by contacting the sample with [0069] a) at least one apoptosis
inhibitor, and/or [0070] b) at least one hypertonic agent, which
stabilizes the cells comprised in the sample, and/or [0071] c) at
least one compound according to formula 1
[0071] ##STR00003## [0072] wherein R1 is a hydrogen residue or an
alkyl residue, preferably a C1-C5 alkyl residue, more preferred a
methyl residue, R2 and R3 are identical or different hydrocarbon
residues with a length of the carbon chain of 1-20 atoms arranged
in a linear or branched manner, and R4 is an oxygen, sulphur or
selenium residue.
[0073] Thereby, the risk is reduced that the extracellular nucleic
acid population is contaminated with intracellular nucleic acids,
in particular fragmented genomic DNA originating from contained
cells, e.g. from damaged or dying cells and/or the degradation of
nucleic acids present in the sample is reduced, respectively
inhibited. This has the effect that the composition of the
extracellular nucleic acid population comprised in said sample is
substantially preserved, respectively stabilized.
[0074] The term "extracellular nucleic acids" or "extracellular
nucleic acid" as used herein, in particular refers to nucleic acids
that are not contained in cells. Respective extracellular nucleic
acids are also often referred to as cell-free nucleic acids. These
terms are used as synonyms herein. Hence, extracellular nucleic
acids usually are present exterior of a cell or exterior of a
plurality of cells within a sample. The term "extracellular nucleic
acids" refers e.g. to extracellular RNA as well as to extracellular
DNA. Examples of typical extracellular nucleic acids that are found
in the cell-free fraction (respectively portion) of biological
samples such as body fluids such as e.g. blood plasma include but
are not limited to mammalian extracellular nucleic acids such as
e.g. extracellular tumor-associated or tumor-derived DNA and/or
RNA, other extracellular disease-related DNA and/or RNA,
epigenetically modified DNA, fetal DNA and/or RNA, small
interfering RNA such as e.g. miRNA and siRNA, and non-mammalian
extracellular nucleic acids such as e.g. viral nucleic acids,
pathogen nucleic acids released into the extracellular nucleic acid
population e.g. from prokaryotes (e.g. bacteria), viruses,
eukaryotic parasites or fungi. According to one embodiment, the
extracellular nucleic acid is obtained from respectively is
comprised in a body fluid as cell-containing biological sample such
as e.g. blood, plasma, serum, saliva, urine, liquor, cerebrospinal
fluid, sputum, lachrymal fluid, sweat, amniotic or lymphatic fluid.
Herein, we refer to extracellular nucleic acids that are obtained
from circulating body fluids as circulating extracellular or
circulating cell-free nucleic acids. According to one embodiment,
the term extracellular nucleic acid in particular refers to
mammalian extracellular nucleic acids, preferably
disease-associated or disease-derived extracellular nucleic acids
such as tumor-associated or tumor-derived extracellular nucleic
acids, extracellular nucleic acids released due to inflammations or
injuries, in particular traumata, extracellular nucleic acids
related to and/or released due to other diseases, or extracellular
nucleic acids derived from a fetus. The term "extracellular nucleic
acids" or "extracellular nucleic acid" as described herein also
refers to extracellular nucleic acids obtained from other samples,
in particular biological samples other than body fluids. Usually,
more than one extracellular nucleic acid is comprised in a sample.
Usually, a sample comprises more than one kind or type of
extracellular nucleic acids. The term "extracellular nucleic acid
population" as used herein in particular refers to the collective
of different extracellular nucleic acids that are comprised in a
cell-containing sample. A cell-containing sample usually comprises
a characteristic and thus unique extracellular nucleic acid
population. Thus, the type, kind and/or the amount of one or more
extracellular nucleic acids comprised in the extracellular nucleic
acid population of a specific sample are important sample
characteristics. As discussed above, it is therefore important to
stabilize and thus to substantially preserve said extracellular
nucleic acid population as its composition and/or the amount of one
or more extracellular nucleic acids comprised in the extracellular
nucleic acid population of a sample, can provide valuable
information in the medical, prognostic or diagnostic field. In
particular, it is important to reduce the contamination and hence
dilution of the extracellular nucleic acid population by
intracellular nucleic acids, in particular by genomic DNA, after
the sample was collected. The substantial preservation of the
extracellular nucleic acid population that can be achieved with the
stabilization technologies according to the invention allows the
population of extracellular nucleic acids within a sample to be
maintained substantially unchanged over the stabilization period as
compared to the population of extracellular nucleic acids at the
moment of sample stabilization. At least, changes in the
extracellular nucleic acid population with respect to the quantity,
the quality and/or the composition of the comprised extracellular
nucleic acids, in particular changes attributable to an increase of
released genomic DNA, are over the stabilization period
considerably reduced (preferably by at least 60%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90% or at least
95%) compared to an unstabilized sample or a corresponding sample
that is e.g. stabilized by EDTA in case of a blood sample or a
sample derived from blood.
[0075] According to a first sub-aspect of the first aspect, at
least one apoptosis inhibitor is used for stabilizing the sample.
As is shown by the provided examples, already the apoptosis
inhibitor alone is effective in stabilizing a cell-containing
sample and to substantially preserve the extracellular nucleic acid
population from changes in its composition in particular arising
from contaminations with fragmented genomic DNA. The sample can be
contacted with the apoptosis inhibitor, e.g. by adding the
apoptosis inhibitor to the sample or vice versa. The at least one
apoptosis inhibitor present in the resulting mixture supports the
stabilization of cells contained in the sample and inhibits the
degradation of nucleic acids comprised in the sample thereby
substantially preserving the extracellular nucleic acid
population.
[0076] The term "apoptosis inhibitor" as used herein in particular
refers to a compound whose presence in a cell-containing biological
sample provides a reduction, prevention and/or inhibition of
apoptotic processes in the cells and/or makes the cells more
resistant to apoptotic stimuli. Apoptosis inhibitors include but
are not limited to proteins, peptides or protein- or peptide-like
molecules, organic and inorganic molecules. Apoptosis inhibitors
include compounds that act as metabolic inhibitors, inhibitors of
nucleic acid degradation respectively nucleic acid pathways, enzyme
inhibitors, in particular caspase inhibitors, calpain inhibitors
and inhibitors of other enzymes involved in apoptotic processes.
Respective apoptosis inhibitors are listed in Table 1. Preferably,
the at least one apoptosis inhibitor that is used for stabilizing
the cell-containing biological sample is selected from the group
consisting of metabolic inhibitors, caspase inhibitors and calpain
inhibitors. Suitable examples for each class are listed in Table 1
in the respective category. Preferably, the apoptosis inhibitor is
cell-permeable.
[0077] It is also within the scope of the present invention to use
a combination of different apoptosis inhibitors, either from the
same or a different class of apoptosis inhibitors, respectively to
use a combination of different apoptosis inhibitors which inhibit
apoptosis either by the same or a different working mechanism.
[0078] In an advantageous embodiment of the present invention, the
apoptosis inhibitor is a caspase inhibitor. Members of the caspase
gene family play a significant role in apoptosis. The substrate
preferences or specificities of individual caspases have been
exploited for the development of peptides that successfully compete
caspase binding. It is possible to generate reversible or
irreversible inhibitors of caspase activation by coupling
caspase-specific peptides to e.g. aldehyde, nitrile or ketone
compounds. E.g. fluoromethyl ketone (FMK) derivatized peptides such
as Z-VAD-FMK act as effective irreversible inhibitors with no added
cytotoxic effects. Inhibitors synthesized with a benzyloxycarbonyl
group (BOC) at the N-terminus and O-methyl side chains exhibit
enhanced cellular permability. Further suitable caspase inhibitors
are synthesized with a phenoxy group at the C-terminus. An example
is Q-VD-OPh which is a cell permeable, irreversible broad-spectrum
caspase inhibitor that is even more effective in preventing
apoptosis than Z-VAD-FMK.
[0079] According to one embodiment, the caspase inhibitor is a
pancaspase inhibitor and thus is a broad spectrum caspase
inhibitor. According to one embodiment, the caspase inhibitor
comprises a modified caspase-specific peptide. Preferably, said
caspase-specific peptide is modified by an aldehyde, nitrile or
ketone compound. According to a preferred embodiment, the caspase
specific peptide is modified preferably at the carboxyl terminus
with an O-Phenoxy or a fluoromethyl ketone (FMK) group. According
to one embodiment, the caspase inhibitor is selected from the group
consisting of Q-VD-OPh and Z-VAD(OMe)-FMK. In one embodiment,
Z-VAD(OMe)-FMK, a pancaspase inhibitor, is used, which is a
competitive irreversible peptide inhibitor and blocks caspase-1
family and caspase-3 family enzymes. In a preferred embodiment,
Q-VD-OPh, which is a broad spectrum inhibitor for caspases, is
used. Q-VD-OPh is cell permeable and inhibits cell death by
apoptosis. Q-VD-OPh is not toxic to cells even at extremely high
concentrations and consists of a carboxy terminal phenoxy group
conjugated to the amino acids valine and aspartate. It is equally
effective in preventing apoptosis mediated by the three major
apoptotic pathways, caspase-9 and caspase-3, caspase-8 and
caspase-10, and caspase-12 (Caserta et al, 2003). Further caspase
inhibitors are listed in Table 1. According to one embodiment, the
caspase inhibitor that is used as apoptosis inhibitor for
stabilizing the cell-containing sample is one which acts upon one
or more caspases located downstream in the intracellular cell death
pathway of the cell, such as caspase-3. In one embodiment of the
present invention the caspase inhibitor is an inhibitor for one or
more caspases selected from the group consisting of caspase-3,
caspase-8, caspase-9, caspase-10 and caspase-12. It is also within
the scope of the present invention to use a combination of caspase
inhibitors.
[0080] The mixture that is obtained after contacting the biological
sample with the at least one apoptosis inhibitor may comprise the
apoptosis inhibitor (or combination of apoptosis inhibitors) in a
concentration selected from the group of at least 0.01 .mu.M, at
least 0.05 .mu.M, at least 0.1 .mu.M, at least 0.5 .mu.M, at least
1 .mu.M, at least 2.5 .mu.M or at least 3.5 .mu.M. Of course, also
higher concentrations can be used. Suitable concentration ranges
for the apoptosis inhibitor(s) when mixed with the cell-containing
biological sample, include but are not limited to 0.01 .mu.M to 100
.mu.M, 0.05 .mu.M to 100 .mu.M, 0.1 .mu.M to 50 .mu.M, 0.5 .mu.M to
50 .mu.M, 1 .mu.M to 40 .mu.M, more preferably 1 .mu.M to 30 .mu.M
or 2.5 .mu.M to 25 .mu.M. The higher concentrations were found to
be more effective, however, good stabilizing results were also
achieved at lower concentrations. Hence, an efficient stabilization
is also achieved at lower concentrations e.g. in a range selected
from 0.1 .mu.M to 10 .mu.M, 0.5 .mu.M to 7.5 .mu.M or 1 .mu.M to 5
.mu.M, in particular if the apoptosis inhibitor is used in
combination with a hypertonic agent (see below). The above
mentioned concentrations apply to the use of a single apoptosis
inhibitor as well as to the use of a combination of caspase
inhibitors. If a combination of caspase inhibitors is used, the
concentration of an individual apoptosis inhibitor that is used in
said mixture of apoptosis inhibitors may also lie below the above
mentioned concentrations, if the overall concentration of the
combination of apoptosis inhibitors fulfils the above mentioned
features. Using a lower concentration that still efficiently
stabilizes the cells and/or reduce the degradation of nucleic acids
in present in the sample has the advantage that the costs for
stabilisation can be lowered. Lower concentrations can be used e.g.
if the apoptosis inhibitor is used in combination with one or more
stabilizers as described herein. The aforementioned concentrations
are in particular suitable when using a caspase inhibitor, in
particular a modified caspase specific peptide such as Q-VD-OPh
and/or Z-VAD(OMe)-FMK as apoptosis inhibitor. The above mentioned
concentrations are e.g. very suitable for stabilizing whole blood,
in particular 10 ml blood. Suitable concentration ranges for other
apoptosis inhibitors and/or for other cell-containing biological
samples can be determined by the skilled person using routine
experiments, e.g. by testing the apoptosis inhibitors, respectively
the different concentrations in the test assays described in the
examples.
[0081] According to one embodiment, the apoptosis inhibitor will,
in an effective amount, decrease or reduce apoptosis in a
cell-containing biological sample by at least 25 percent, at least
30 percent, at least 40 percent, at least 50 percent, preferably,
by at least 75 percent, more preferably, by at least 85 percent as
compared to a control sample which does not contain a respective
apoptosis inhibitor.
[0082] According to a second sub-aspect of the first aspect of the
present invention, at least one hypertonic agent is used for
stabilizing the sample, wherein the used hypertonic agent
stabilizes cells comprised in the sample. As is shown by the
provided examples, already the hypertonic agent alone is effective
in stabilizing a cell-containing sample and substantially
preserving the composition of the extracellular nucleic acid
population comprised therein. The hypertonic agent induces cell
shrinking by mild hypertonic effects (osmosis), thereby increasing
the cell stability. Therefore, the cells are less prone to e.g.
mechanically induced cell damage. The sample can be contacted with
the hypertonic agent, e.g. by adding the hypertonic agent to the
sample or vice versa. The hypertonic agent present in the resulting
mixture in particular is suitable for stabilizing cells contained
in the sample, thereby reducing the amount of intracellular nucleic
acids, in particular genomic DNA that is released from damaged
cells. Thereby, the extracellular nucleic acid population is
substantially preserved and the risk of contaminating respectively
diluting the extracellular nucleic acids with intracellular nucleic
acids, in particular genomic DNA, is reduced.
[0083] According to one embodiment, the hypertonic agent is
sufficiently osmotically active to induce cell shrinking (the cells
release water), however, without damaging the cells i.e. without
inducing or promoting cell lysis, respectively cell rupture. Hence,
the hypertonic agent preferably has a mild osmotic effect.
Furthermore, it is desirous that interactions between the
hypertonic agent and the sample are predominantly limited to the
cell stabilization effect basically in order to avoid unwanted side
effects. Thus, according to one embodiment, an uncharged hypertonic
agent is used. Using an uncharged hypertonic agent has the
advantage that even though the cells shrink respectively are
stabilized due to the osmotic effect of the hypertonic agent,
interactions between the hypertonic agent and other compounds
comprised in the sample are limited compared to the use of a
charged hypertonic agent.
[0084] According to an advantageous embodiment, the hypertonic
agent is a hydroxylated organic compound and accordingly, carries
at least one hydroxyl group. According to one embodiment, the
hydroxylated organic compound comprises at least two hydroxyl
groups. According to one embodiment, the hydroxylated organic
compound is a polyol. According to one embodiment, the polyol
comprises 2 to 10 hydroxyl groups, preferably 3 to 8 hydroxyl
groups. The hydroxylated organic compound may comprise 2 to 12
carbon atoms, preferably 3 to 8 and can be a cyclic or linear
molecule, branched or un-branched; it can be saturated or
unsaturated; aromatic or non-aromatic. According to one embodiment,
the hydroxylated organic compound is a hydroxy-carbonyl compound. A
hydroxy-carbonyl compound is a compound possessing one or more
hydroxy (OH) groups and one or more carbonyl groups. Hydroxylated
organic compounds may include but are not limited to hydroxylated
ketone compounds and carbohydrates, or compounds derived therefrom.
According to one embodiment, the hydroxylated organic compound is a
polyalcohol, in particular a sugar alcohol. Hence, hydroxylated
organic compounds include but are not limited to carbohydrates such
as glucose, raffinose, succrose, fructose, alpha-d-lactose
monohydrate, inositol, maltitol, mannitol, dihydroxyacetone,
alcohols such as glycerol, erythritol, mannitol, sorbitol,
volemitol, or sugar alcohols. Suitable examples are also listed in
the table below. It is also within the scope of the present
invention to use combinations of respective hydroxylated organic
compounds.
TABLE-US-00001 Chemical Formula IUPAC Name Common Name Polyols,
e.g. C.sub.3H.sub.5(OH).sub.3 Propane-1,2,3-triol Glycerin
C.sub.4H.sub.6(OH).sub.4 Butane-1,2,3,4-tetraol Erythritol
C.sub.5H.sub.7(OH).sub.5 Pentane-1,2,3,4,5-pentol Xylitol,
Arabitol, Ribitol C.sub.6H.sub.8(OH).sub.6 Hexane-1,2,3,4,5,6-hexol
Mannitol, Sorbitol, Dulcitol, Iditol C.sub.7H.sub.9(OH).sub.7
Heptane-1,2,3,4,5,6,7- Volemitol heptol Alicyclic and sugar
alcohols, e.g. C.sub.6H.sub.6(OH).sub.6 Cyclohexane-1,2,3,4,5,6-
Inositol geksol C.sub.12H.sub.24O.sub.11
1-O-.alpha.-D-Glucopyranosyl-D- Isomalt mannitol
C.sub.12H.sub.24O.sub.11 4-O-.alpha.-D-Glucopyranosyl-D- Maltitol
glucitol C.sub.12H.sub.24O.sub.11 4-O-.alpha.-D-Galactopyranosyl-
Lactitol D-glucitol
[0085] According to one embodiment, the polyols and sugar alcohols
listed above may be replaced by alcohols with less hydroxyl groups
(e.g., hexane-1,2,3,4,5-pentol, pentane-1,2,3,4-tetraol). According
to one embodiment, the hydroxylated organic compound is no alcohol
having 1 to carbon atoms and carrying only one hydroxyl group.
According to one embodiment, alcohols with only one hydroxyl group
are excluded as hydroxylated organic compound. The hydroxylated
organic compound that can be used as stabilizer according to the
present invention preferably is water-soluble and non-toxic to the
cells comprised in the biological sample to be stabilized.
Preferably, the hydroxylated organic compound does not induce or
support the lysis of the cells contained in the biological sample
and accordingly, preferably does not function as a detergent or as
cell membrane dissolving agent. A suitable hydroxylated organic
compound according to the present invention achieves a stabilizing
effect of the cell-containing sample by improving the preservation
of the composition of the extracellular nucleic acid population as
can be e.g. tested by the assays described in the example
section.
[0086] Adding a hydroxylated organic compound to a cell-containing
biological sample such as e.g. whole blood, increases the
concentration of said hydroxylated organic compound in the
cell-free portion respectively fraction (e.g. the blood plasma) and
thus forces blood cells to release water into the plasma as a
result of an osmotic (hypertonic) effect. According to one
embodiment, a hydroxylated organic compound is used which is
closely related to a product of the cell metabolism but preferably
can not be utilized by the cells.
[0087] According to a preferred embodiment, cells contained in the
biological sample are essentially impermeable for the hypertonic
agent that is used for stabilization. Thus, the hypertonic agent,
which preferably is a hydroxylated organic compound as described in
detail above, is essentially cell impermeable. Essentially cell
impermeable in this respect in particular means that the
concentration of the hypertonic agent, which preferably is a
hydroxylated organic compound, is substantially higher in the
extracellular portion of the sample than inside the cells contained
in the biological sample that is stabilized according to the
teachings of the present invention. According to a preferred
embodiment, the hypertonic agent, which preferably is a
hydroxylated organic compound, is non-toxic, so that the cell
viability is not compromised. This is preferred to avoid disturbing
influences on the cell metabolism.
[0088] According to one embodiment, the hypertonic agent is
dihydroxyacetone (DHA). DHA is a carbohydrate and usually serves as
tanning substance in self-tanning lotions. As is demonstrated by
the examples, DHA surprisingly has a remarkable stabilizing effect
on cell-containing biological samples, in particular whole blood
samples and samples derived from whole blood such as blood plasma
or serum. DHA does naturally not occur in mammalian cells except
for the phosphoric acid ester of DHA, dihydroxyacetone-phosphat, an
intermediate product of glycolysis. Thus DHA is not expected to be
actively transported or to diffuse into blood cells. According to
one embodiment, the hypertonic agent is not
dihydroxyaceton-phosphate.
[0089] The mixture that is obtained when contacting the
cell-containing biological sample with the at least one hypertonic
agent may comprise the hypertonic agent or mixture of hypertonic
agents in a concentration of at least 0.05M, preferably 0.1M,
preferably at least 0.2M, more preferred at least 0.25M. Of course,
also higher concentrations can be used. Suitable concentration
ranges for the hypertonic agent can be selected from 0.05M to 2M,
0.1M to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M or 0.1M to 0.6M.
Respective concentrations are particularly suitable when using a
hydroxylated organic compound, e.g. a carbohydrate such as
dihydroxyacetone as hypertonic agent. The above mentioned
concentrations are e.g. very suitable for stabilizing whole blood,
in particular 10 ml blood. Suitable concentration ranges for other
hypertonic agents and/or other cell-containing biological samples
can also be determined by the skilled person using routine
experiments, e.g. by testing the hypertonic agents, respectively
different concentrations thereof in the test assays described in
the examples.
[0090] According to a third sub-aspect of the first aspect of the
present invention, for stabilizing the extracellular nucleic acid
population in a cell containing sample, at least one compound
according to formula 1 is used
##STR00004##
wherein R1 is a hydrogen residue or an alkyl residue, preferably a
C1-C5 alkyl residue, more preferred a methyl residue, R2 and R3 are
identical or different hydrocarbon residues with a length of the
carbon chain of 1-20 atoms arranged in a linear or branched manner,
and R4 is an oxygen, sulphur or selenium residue.
[0091] As is shown by the provided examples, a compound according
to formula 1 described above is effective in achieving a remarkable
stabilizing effect and in substantially preserving the composition
of the extracellular nucleic acid population in the stabilized
sample. Also a mixture of one or more compounds according to
formula 1 can be used for stabilization.
[0092] The hydrocarbon residues R2 and/or R3 can be selected
independently of one another from the group comprising alkyl,
including short chain alkyl and long-chain alkyl, alkenyl, alkoxy,
long-chain alkoxy, cycloalkyl, aryl, haloalkyl, alkylsilyl,
alkylsilyloxy, alkylene, alkenediyl, arylene, carboxylates and
carbonyl. General groups, for instance alkyl, alkoxy, aryl etc. are
claimed and described in the description and the claims.
Preferably, the following groups are used within the generally
described groups within the scope of the present invention: [0093]
(1) alkyl: preferably short chain alkyls, in particular linear and
branched C1-C5 alkyls or long-chain alkyls: linear and branched
C5-C20 alkyls; [0094] (2) alkenyl: preferably C2-C6 alkenyl; [0095]
(3) cycloalkyl: preferably C3-C8 cycloalkyl; [0096] (4) alkoxy:
preferably C1-C6 alkoxy; [0097] (5) long-chain alkoxy: preferably
linear and branched C5-C20 alkoxy; [0098] (6) alkylenes: preferably
a divalent linear or branched aliphatic, cycloaliphatic or aromatic
hydrocarbon residue with 2 to 18 carbon atoms optionally containing
heteroatoms, e.g. selected from the group comprising: methylene;
1,1-ethylene; 1,1-propylidene; 1,2-propylene; 1,3-propylene;
2,2-propylidene; butan-2-ol-1,4-diyl; propan-2-ol-1,3-diyl;
1,4-butylene; 1,4-pentylene; 1,6-hexylene; 1,7-heptylene;
1,8-octylene; 1,9-nonylene; 1,10-decylene; 1,11-undecylene;
1,12-docedylene; cyclohexane-1,1-diyl; cyclohexane-1,2-diyl;
cyclohexane-1,3-diyl; cyclohexane-1,4-diyl; cyclopentane-1,1-diyl;
cyclopentane-1,2-diyl; and cyclopentane-1,3-diyl; [0099] (7)
alkenediyl: preferably selected from the group comprising:
1,2-propenediyl; 1,2-butenediyl; 2,3-butenediyl; 1,2-pentenediyl;
2,3-pentenediyl; 1,2-hexenediyl; 2,3-hexenediyl; and
3,4-hexenediyl; [0100] (8) alkynediyl: is equal to --C.ident.C;
[0101] (9) aryl: preferably selected from aromatics with a
molecular weight below 300 Da; [0102] (10) arylenes: preferably
selected from the group comprising: 1,2-phenylene; 1,3-phenylene;
1,4-phenylene; 1,2-naphtthalenylene; 1,3-naphtthalenylene;
1,4-naphtthalenylene; 2,3-naphtthalenylene;
1-hydroxy-2,3-phenylene; 1-hydroxy-2,4-phenylene;
1-hydroxy-2,5-phenylene; 1-hydroxy-2,6-phenylene; [0103] (11)
carboxylate: preferably the group --C(O)OR, where R is selected
from: hydrogen; C1-C6 alkyl; phenyl; C1-C6 alkyl-C6H5; Li; Na; K;
Cs; Mg; Ca; [0104] (12) carbonyl: preferably the group --C(O)R,
where R is selected from: hydrogen; C1-C6 alkyl; phenyl; C1-C6
alkyl-C6H5 and amine (resulting in an amide) selected from the
group: --NR'2, where each R' is selected independently from:
hydrogen; C1-C6 alkyl; C1-C6 alkyl-C6H5 and phenyl, where, if both
Rs represent C1-C6 alkyl they can form an NC3 to NC5 heterocyclic
ring with alkyl substituents of the ring forming the other alkyl
chain; [0105] (13) alkylsilyl: preferably the group --SiR1R2R3,
where R1, R2 and R3 are selected independently of one another from:
hydrogen; alkyl; long-chain alkyl; phenyl; cycloalkyl; haloalkyl;
alkoxy; long-chain alkoxy; [0106] (14) alkylsilyloxy: preferably
the group --O--SiR1R2R3, where R1, R2 and R3 are selected
independently of one another from: hydrogen; alkyl; long-chain
alkyl; phenyl; cycloalkyl; haloalkyl; alkoxy; long-chain
alkoxy.
[0107] The chain length n of R2 and/or R3 can in particular have
the values 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 and 20. Preferably R2 and R3 have a length of the carbon
chain of 1-10. In this case the chain length n can in particular
have the values 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Preferably, R2
and R3 have a length of the carbon chain of 1-5 and in this case
the chain length can in particular have the values 1, 2, 3, 4 and
5. Particularly preferred is a chain length of 1 or 2 for R2 and
R3.
[0108] The chain length n of R1 preferably has the value 1,2,3,4 or
5. Particularly preferred is a chain length of 1 or 2 for R1.
[0109] R4 preferably is oxygen.
[0110] According to a preferred embodiment, the compound according
to formula 1 is a N,N-dialkyl-carboxylic acid amide. Preferred R1,
R2, R3 and R4 groups are described above. According to one
embodiment, the compound is selected from the group consisting of
N,N-dimethylacetamide; N,N-diethylacetamide; N,N-dimethylformamide
and N,N-diethylformamide. Also suitable are N,N-dialkylpropanamides
such as N,N-dimethylpropanamide as is shown in the examples.
Preferably, the substance according to formula 1 is
N,N-dimethlylacetamide (DMAA). The structural formulae of the
preferred compounds are as follows:
##STR00005##
[0111] Also suitable are the respective thio analogues, which
comprise sulphur instead of oxygen as R4.
[0112] The mixture that is obtained when contacting the
cell-containing biological sample with a compound according to
formula 1 or a mixture of respective compounds may comprise said
compound or mixture of compounds in a final concentration of at
least 0.1%, at least 0.5%, at least 0.75%, at least 1%, at least
1.25% or at least 1.5%. A suitable concentration range includes but
is not limited to 0.1% up to 50%. Preferred concentration ranges
can be selected from the group consisting of 0.1% to 30%, 0.1% to
20%, 0.1% to 15%, 0.1% to 10%, 0.1% to 7.5%, 0.1% to 5%, 1% to 30%,
1% to 20%, 1% to 15%, 1% to 10%, 1% to 7.5%, 1% to 5%; 1.25% to
30%, 1.25% to 20%, 1.25% to 15%, 1.25% to 10%, 1.25% to 7.5%, 1.25%
to 5%; 1.5% to 30%, 1.5% to 20%, 1.5% to 15%, 1.5% to 10%, 1.5% to
7.5% and 1.5% to 5%. Respective concentrations are particularly
suitable when using a N,N-dialkyl-carboxylic acid amide, e.g.
N,N-dimethylacetamide, N,N-diethylacetamide, N,N-diethylformamide
or N,N-diemethylformamide or N,N-dimethylpropanamide as stabilizing
agent. The above mentioned concentrations are e.g. very suitable
for stabilizing whole blood or blood products such as plasma.
Suitable concentration ranges for other compounds according to
formula 1 and/or other cell-containing biological samples can also
be determined by the skilled person using routine experiments, e.g.
by testing the compound, respectively different concentrations
thereof in the test assays described in the examples.
[0113] Preferably, the compound according to formula 1 is used in
combination with a chelating agent for stabilizing the cell
containing sample. In particular, a chelating agent can be used as
anticoagulant when stabilizing a blood sample or a sample derived
from blood such as e.g. plasma or serum. Suitable chelating agents
and concentration ranges are provided below.
[0114] According to a preferred fourth sub-aspect, a method
suitable for stabilizing a cell-containing sample, preferably a
blood sample is provided, wherein said method comprises contacting
the sample with [0115] a) at least one apoptosis inhibitor, and
[0116] b) at least one hypertonic agent, which stabilizes the cells
comprised in the sample.
[0117] Thus, according to this preferred embodiment, the apoptosis
inhibitor and the hypertonic agent, which both alone are already
effective in stabilizing a cell-containing sample (see above and
examples), are used in combination. Thereby, the stabilization
effect can be increased and/or the concentration of the individual
components (the apoptosis inhibitor and/or the hypertonic agent)
may also be reduced while still efficiently preserving the
extracellular nucleic acid population in the sample, and in
particular avoiding, respectively reducing the contamination by
intracellular nucleic acids in particular fragmented genomic DNA
that is released from damaged or decaying cells contained in the
sample. As is shown in the examples, using a respective combination
is particularly effective in stabilizing a cell-containing sample,
even very complex samples such as a whole blood sample. It is also
within the scope of the present invention to use a mixture of
different apoptosis inhibitors in combination with different
hypertonic agents. Suitable and preferred embodiments of the
apoptosis inhibitor and the hypertonic agent as well as suitable
and preferred concentrations of the respective agents suitable for
achieving an efficient stabilization of the sample are described in
detail above in conjunction with the embodiments, wherein either an
apoptosis inhibitor or a hypertonic agent is used to stabilize the
cell-containing biological sample. It is referred to the above
disclosure which also applies to the embodiment, wherein an
apoptosis inhibitor is used in combination with a hypertonic agent.
Preferably, at least one caspase inhibitor, preferably a modified
caspase specific peptide, preferably modified at the C-terminus
with an O-phenoxy group such as Q-VD-OPh, is used in combination
with at least one hydroxylated organic compound, e.g. a
carbohydrate, such as dihydroxyacetone or a polyol, as hypertonic
agent. As is demonstrated by the examples, a respective combination
is remarkably effective in stabilizing a cell-containing biological
sample, in particular a whole blood sample, at room temperature for
more than 3 days and even for 6 days.
[0118] According to one embodiment, a combination of stabilizing
agents is used which comprises at least one apoptosis inhibitor, at
least one hypertonic agent and/or at least one compound according
to formula 1 as defined above. Examples of respective combinations
include (1) a combination of at least one apoptosis inhibitor and
at least one compound according to formula 1 as defined above, (2)
a combination of at least one hypertonic agent and at least one
compound according to formula 1 as defined above or (3) a
combination of all three stabilizing agents, i.e. at least one
apoptosis inhibitor, at least one hypertonic agent and at least one
compound according to formula 1 as defined above. A respective
combination may also comprise additional additives that enhance the
stabilizing effect such as e.g. anticoagulants and chelating
agents. According to one embodiment, the combination of stabilizing
agents comprises a caspase inhibitor and an anticoagulant,
preferably a chelating agent such as EDTA. Respective combinations
can be according to a fifth sub-aspect advantageously used in a
method suitable for stabilizing an extracellular nucleic acid
population comprised in a cell-containing sample according to the
first aspect of the present invention. The stabilizing effect
observed with combinations of stabilizing agents is stronger than
the effect observed for any of the individual stabilizing agents
when used alone and/or allows to use lower concentrations, thereby
making combinatorial use of stabilizing agents an attractive
option. Suitable and preferred embodiments of the apoptosis
inhibitor, the hypertonic agent and the compound according to
formula 1 defines above as well as suitable and preferred
concentrations of the respective agents suitable for achieving an
efficient stabilization of the sample are described in detail above
in conjunction with the embodiments, wherein either an apoptosis
inhibitor, a hypertonic agent or a compound according to formula 1
is used to stabilize the cell-containing biological sample.
[0119] As discussed in the background of the invention,
extracellular nucleic acids are usually not present "naked" in the
sample but are e.g. stabilized to a certain extent by being
released protected in complexes or by being contained in vesicles
and the like. This has the effect that extracellular nucleic acids
are already to a certain extent stabilized by nature and thus, are
usually not degraded rapidly by nucleases in cell-containing
samples such as whole blood, plasma or serum. Thus, when intending
to stabilize extracellular nucleic acids that are comprised in a
biological sample, one of the primary problems is the dilution,
respectively the contamination of the extracellular nucleic acid
population by intracellular nucleic acids, in particular fragmented
genomic DNA, that originates from damaged or dying cells that are
contained in the sample. This also poses a problem when processing
cell-depleted samples such as plasma or serum (which are sometimes
also describes as being "cell-free" even though they may comprise
minor amounts of cells). The stabilization technology according to
the present invention is of particular advantage in this respect
because it not only substantially preserves the extracellular
nucleic acids present in the sample and e.g. inhibits degradation
of the comprised extracellular nucleic acids (preferably at least
by 60%, at least by 70%, at least by 75%, at least by 80%, at least
by 85%, at least by 90% or most preferably at least by 95% over the
stabilization period compared to an unstabilized sample or an EDTA
stabilized sample) but furthermore, efficiently reduces the release
of genomic DNA from cells contained in the sample and/or reduces
the fragmentation of respective genomic DNA. According to one
embodiment, using the apoptosis inhibitor, the hypertonic agent
and/or the compound according to formula 1 for stabilizing the
cell-containing sample according to the teachings of the present
invention has the effect that the increase of DNA that results from
a release of DNA from cells contained in the sample is reduced
compared to a non-stabilized sample. According to one embodiment,
said release of genomic DNA is reduced by at least 3-fold, at least
4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least
10-fold, at least 12-fold, at least 15-fold, at least 17-fold or at
least 20-fold over the stabilization period compared to the
non-stabilized sample or a corresponding sample that is stabilized
with EDTA (in particular in case of a blood sample or a sample
derived from blood such as plasma or serum). According to one
embodiment, said release of genomic DNA is reduced by at least 60%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90% or at least 95% over the stabilization period compared to the
non-stabilized sample or a corresponding sample that is stabilized
with EDTA (in particular in case of a blood sample or a sample
derived from blood such as plasma or serum). The release of DNA can
be determined e.g. by quantifying the ribosomal 18S DNA as is
described herein in the example section. E.g. standard EDTA
stabilized blood samples show a 40-fold increase of DNA determined
e.g. at day 6 of storage at room temperature in a respective assay
(see FIG. 2b). The stabilization achievable with the teachings of
the present invention remarkably reduces this release of DNA even
down to e.g. a maximum of 4-fold. Thus, the extracellular nucleic
acid population contained in the sample is considerably stabilized
compared to samples stabilized in standard EDTA tubes. Thus,
according to one embodiment, the stabilization effect that is
achieved with the apoptosis inhibitor, the hypertonic agent and/or
the compound according to formula 1 as taught by the present
invention results in that the release of DNA from cells contained
in the sample is at least reduced to a maximum of 10-fold,
preferably 7-fold, more preferably 5-fold and most preferably is at
least reduced to a maximum of 4-fold, as is e.g. determinable in
the 18S DNA assay described in the examples. As is shown by the
examples, an effective stabilization of the extracellular nucleic
acid population is achievable for a period of at least up to 6
days. During a shorter storage of the samples, e.g. up to three
days, the DNA release can be reduced at least to a maximum of
two-fold as e.g. determinable in the 18S DNA assay described in the
examples. Thus, the DNA release can be reduced to 2fold or less up
to three days of storage when using the stabilizing methods
according to the present invention. This is a remarkable
improvement in the stabilization of the extracellular nucleic acid
population compared to the prior art methods. This significantly
enhances the accuracy of any subsequent tests. In certain cases,
for example if the sample material has to be transported for long
distances or stored for longer periods e.g. at room temperature (as
can be e.g. the case in certain countries), the process according
to the invention makes it possible for the first time for these
tests to be carried out after such a period of time. However, of
course, the samples may also be further processed earlier, if
desired. It is not necessary to make use of the full achievable
stabilization period. The stabilization that is achieved with the
present invention reduces variations in the extracellular nucleic
acid population that may result from a different
handling/processing of the samples (e.g. storage conditions and
periods) after they were collected. This greatly improves the
standardization of handling and molecular analysis.
[0120] Further additives may be used in addition to the apoptosis
inhibitor, the hypertonic agent and/or the compound according to
formula 1 as defined above in order to further stabilize the
cell-containing sample. The selection of suitable additives that
may also contribute to the stabilization effect may also depend on
the type of cell-containing sample to be stabilized. E.g. when
processing whole blood as cell-containing biological sample, it is
advantageous and also common to include an anticoagulant e.g.
selected from the group consisting of heparin, ethylenediamine
tetraacetic acid, citrate, oxalate, and any combination thereof. In
an advantageous embodiment, the anticoagulant is a chelating agent.
A chelating agent is an organic compound that is capable of forming
coordinate bonds with metals through two or more atoms of the
organic compound. Chelating agents according to the present
invention include, but are not limited to
diethylenetriaminepentaacetic acid (DTPA),
ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol
tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA).
According to a preferred embodiment, EDTA is used. As used herein,
the term "EDTA" indicates inter alia the EDTA portion of an EDTA
compound such as, for example, K.sub.2EDTA, K.sub.3EDTA or
Na.sub.2EDTA. Using a chelating agent such as EDTA also has the
advantageous effect that nucleases such as DNases are inhibited,
thereby e.g. preventing a degradation of extracellular DNA by
DNases. Furthermore, it was found by the inventors that EDTA
used/added in higher concentrations is capable of reducing the
release of intracellular nucleic acids, in particular genomic DNA
from the cells thereby supporting the stabilizing effect that is
achieved by the apoptosis inhibitor, the hypertonic agent and/or
the at least one compound according to formula 1. However, EDTA
alone is not capable of efficiently inhibiting the fragmentation of
e.g. genomic DNA that is released from the cells contained in the
sample. Thus, EDTA does not achieve a sufficient stabilization
effect. But used in combination with the teachings of the present
invention, in particular in combination with the apoptosis
inhibitor, in particular the caspase inhibitor, it can further
improve the stabilization for the above discussed reasons.
Furthermore, it also appears to increase the chemical stability of
RNA. According to one embodiment, the concentration of the
chelating agent, preferably EDTA, in the biological sample that is
mixed with one or more of the stabilizing compounds described above
is in the range selected from the group consisting of 0.05 mM to
100 mM, 0.05 mM to 50 mM, 0.1 mM to 30 mM, 1 mM to 20 mM and 2 mM
to 15 mM after the contacting step. Respective concentrations are
particularly effective when stabilising blood, plasma and/or serum
samples, in particular 10 ml blood samples.
[0121] Additional additives can also be used in order to further
support the stabilization of the cell-containing sample,
respectively support the preservation of the extracellular nucleic
acid population. Examples of respective additives include but are
not limited to nuclease inhibitors, in particular RNase and DNase
inhibiting compounds. Examples of RNase inhibitors include but are
not limited to anti-nuclease antibodies or
ribonucleoside-vanadyl-complexes. When choosing a respective
further additive, care should be taken not to compromise and/or
counteract the stabilizing effect of the apoptosis inhibitor, the
hypertonic agent and/or the compound according to formula 1. Thus,
no additives should be used in concentrations that result in or
support the lysis and/or degradation of the cells contained in the
biological sample and/or which support the degradation of the
nucleic acids contained in the cell-free fraction of the biological
sample.
[0122] In an advantageous embodiment of the present invention, the
cell-containing biological sample, which preferably is a blood
sample or a sample derived from blood such as plasma or serum, is
contacted with: [0123] a) at least one caspase inhibitor as an
apoptosis inhibitor, preferably with Q-VD-OPh, preferably in a
concentration range of 1 .mu.M to 30 .mu.M; [0124] b) optionally at
least one hydroxylated organic compound such as dihydroxyacetone as
hypertonic agent, preferably in a concentration range of 0.1 M to
0.6M; and [0125] c) optionally at least one compound according to
formula 1 defined above (preferred embodiments and concentrations
are described above) and/or [0126] d) a further additive,
preferably a chelating agent preferably in a concentration range of
4 mM to 50 mM, preferably 4 mM to 20 mM, most preferably EDTA.
[0127] The components of the stabilizing composition can be
comprised, respectively dissolved in a buffer, e.g. a biological
buffer such as MOPS, TRIS, PBS and the like.
[0128] The apoptosis inhibitor, the hypertonic agent and/or the
compound according to formula 1 as defined above as well as the
optionally present further additives can be e.g. present in a
device, preferably a container, for collecting the sample or can be
added to a respective collection device immediately prior to
collection of the biological sample; or can be added to the
collection device immediately after the sample was collected
therein. It is also within the scope of the present invention to
add the stabilizing agent(s) and optionally, the further
additive(s) separately to the cell containing biological sample.
However, for the ease of handling, it is preferred that the one or
more stabilizing agents and optionally the further additives are
provided in one composition. Furthermore, in an advantageous
embodiment, the apoptosis inhibitor, the hypertonic agent and/or
the compound according to formula 1 as described above and
optionally the further additive(s) are present in the collection
device prior to adding the sample. This ensures that the
cell-containing biological sample is immediately stabilized upon
contact with the stabilizing agent(s). The stabilisation agent(s)
are present in the container in an amount effective to provide the
stabilisation of the amount of cell containing sample to be
collected, respectively comprised in said container. As described,
the sample can be mixed with the stabilization agent(s) directly
after and/or during collection of the sample thereby providing a
stabilized sample.
[0129] Preferably, the sample is mixed with the stabilization
agent(s) directly after and/or during the collection of the sample.
Therefore, preferably, the stabilization agent(s) and additives
described above are provided in form of a stabilizing composition.
Preferably, said stabilizing composition is provided in liquid
form. It can be e.g. pre-filled in the sample collection device so
that the sample is immediately stabilized during collection.
According to one embodiment, the stabilizing composition is
contacted with the cell-containing sample in a volumetric ratio
selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to
1:5. It is a particular advantage of the teachings of the present
invention that stabilization of a large sample volume can be
achieved with a small volume of the stabilizing composition.
Therefore, preferably, the ratio of stabilizing composition to
sample lies in a range from 1:2 to 1:7, more preferred 1:3 to
1:5.
[0130] The term "cell-containing sample" as used herein, in
particular refers to a sample which comprises at least one cell.
The cell-containing sample may comprise at least two, at least 10,
at least 50, at least 100, at least 250, at least 500, at least
1000, at least 1500, at least 2000 or at least 5000 cells.
Furthermore, also cell-containing samples comprising considerably
more cells are encompassed by said term and can be stabilized with
the teachings according to the present invention. However, the term
"cell-containing sample" also refers to and thus encompasses
cell-depleted samples, including cell-depleted samples that are
commonly referred to as "cell-free" such as e.g. blood plasma as
respective samples often include residual cells. At least, it can
often not be fully excluded that even so-called "cell-free" samples
such as blood plasma comprise residual amounts of cells which
accordingly, pose a risk that the extracellular nucleic acid
population becomes contaminated with intracellular nucleic acids
released from said residual cells. Therefore, respective
cell-depleted and "cell-free" samples are according to one
embodiment also encompassed by the term "cell-containing sample".
Thus, the "cell-containing sample" may comprise large amounts of
cells, as is the case e.g. with whole blood, but may also only
comprise merely minor amounts of cells. Hence, the term "cell
containing sample" also encompasses samples that may only be
suspected of or pose a risk of containing cells. As discussed
above, also with respect to biological samples which only comprise
minor, respectively residual amounts of cells such as e.g. blood
plasma (blood plasma contains--depending on the preparation
method--usually small residual amounts of cells, even though it is
commonly referred to as being cell-free), the method according to
the present invention has considerable advantages as these residual
cells may also result in a undesired contamination of the comprised
extracellular nucleic acids. Using the stabilizing technology of
the present invention also ensures that respective samples which
only comprise residual amounts of cells or are merely suspected of
or pose a risk of residual amounts of cells, are efficiently
stabilized as is also described in detail above. Using the
stabilizing method according to the present invention has the
advantage that irrespective of the composition of the sample and
the number of cells contained therein, the extracellular nucleic
acid population contained therein is substantially preserved,
respectively stabilized, thereby allowing for standardizing the
subsequent isolation and/or analysis of the contained extracellular
nucleic acids.
[0131] According to one embodiment, the cell-containing biological
sample is selected from the group consisting of whole blood,
samples derived from blood, plasma, serum, sputum, lachrymal fluid,
lymphatic fluid, urine, sweat, liquor, cerebrospinal fluid,
ascites, milk, stool, bronchial lavage, saliva, amniotic fluid,
nasal secretions, vaginal secretions, semen/seminal fluid, wound
secretions, and cell culture supernatants and supernatants obtained
from other swab samples. According to one embodiment, the
cell-containing biological sample is a body fluid, a body secretion
or body excretion, preferably a body fluid, most preferably whole
blood, plasma or serum. The cell-containing biological sample
comprises extracellular nucleic acids. According to another
embodiment, the cell-containing biological sample is a non-fluid
sample derived from a human or animal, such as e.g. stool, tissue
or a biopsy sample. Other examples of cell-containing biological
samples that can be stabilized with the method according to the
present invention include but are not limited to biological samples
cell suspensions, cell cultures, supernatant of cell cultures and
the like, which comprise extracellular nucleic acids.
[0132] As is described above and as is demonstrated by the
examples, using the methods of the present invention allows for
stabilizing the cell-containing sample without refrigeration or
freezing for a prolonged period of time period. Thus, the samples
can be kept at room temperature or even at elevated temperatures
e.g. up to 30.degree. C. or up to 40.degree. C. According to one
embodiment, a stabilization effect is achieved for at least two
days, preferably at least three days; more preferred at least one
day to six days, most preferred for at least one day to at least
seven days at room temperature. As is shown in the examples, the
samples that were stabilized according to the method of the present
invention were not substantially compromised when stored for 3 days
at room temperature. Even during longer storages for up to 6 or
even 7 days at room temperature the extracellular nucleic acid
population was substantially more stabilized compared to
non-stabilized samples or e.g. compared to samples that were
stabilized using standard method such as an EDTA treatment. Even
though the stabilization effect may decrease over time, it is still
sufficient to preserve the composition of the extracellular nucleic
acid population to allow the analysis and/or further processing.
Thus, samples that were stabilized according to the methods of the
present invention were still suitable for isolating and optionally
analysing the extracellular nucleic acids contained therein even
after longer storage at room temperature. Thus, as the samples were
not compromised in particular when using the preferred combination
of stabilisation agents, even longer storage/shipping times are
conceivable. However, usually, longer periods are not necessary, as
the regular storage and e.g. shipping time to the laboratory,
wherein the nucleic acid isolation and optionally analysis is
performed, usually does not exceed 6 or 7 days, but usually is even
completed after two or three days. As is shown in the examples, the
stabilisation efficiency is particularly good during this time
period. However, the extraordinary long stabilisation times and
stabilisation efficiencies that are achievable with the method
according to the present invention provides an important safety
factor.
[0133] The methods and also the subsequently described compositions
according to the present invention allow the stabilization also of
large volumes of biological samples with small volumes of added
substances because the additives that are used according to the
teachings of the present invention are highly active. This is an
important advantage because the size/volume of the sample poses
considerable restrains on the subsequent isolation procedure in
particular when intending to use automated processes for isolating
the extracellular nucleic acids contained in the samples.
Furthermore, one has to consider that extracellular nucleic acids
are often only comprised in small amounts in the contained sample.
Thus, processing larger volumes of a cell-containing sample such as
e.g. a blood sample has the advantage that more circulating nucleic
acids can be isolated from the sample and thus are available for a
subsequent analysis.
[0134] The stabilization of the biological sample may either be
followed directly by techniques for analysing nucleic acids, or the
nucleic acids may be purified from the sample. Hence, the sample
that was stabilized according to the method of the present
invention can be analysed in a nucleic acid analytic and/or
detection method and or may be further processed. E.g.
extracellular nucleic acid can be isolated from the stabilized
sample and can then be analysed in a nucleic acid analytic and/or
detection method or may be further processed.
[0135] Furthermore, according to a second aspect, a method for
isolating extracellular nucleic acids from a cell-containing
biological sample is provided, wherein said method comprises the
steps of: [0136] a) stabilizing the extracellular nucleic acid
population comprised in a cell-containing sample according to the
method defined in the first aspect of the present invention; [0137]
b) isolating extracellular nucleic acids.
[0138] As discussed above, the stabilization according to the
present invention has the effect that the extracellular nucleic
acid population contained in the sample is substantially preserved
in the state it had shown at the time the biological sample was
obtained, respectively drawn. In particular, the usually observed
high increase in nucleic acids that results from intracellular
nucleic acids, in particular genomic DNA, more specifically
fragmented genomic DNA, released from damaged or dying cells is
efficiently reduced as is demonstrated in the examples. Therefore,
the extracellular nucleic acids obtained from a respectively
stabilized sample comprise fewer contaminations with intracellular
nucleic acids originating from degraded or dying cells comprised in
the sample and in particular comprise less amounts of fragmented
genomic DNA compared to non-stabilized samples. Furthermore, the
unique stabilization step allows to increase the amount of
recoverable extracellular nucleic acids. The stabilization method
according to the present invention can be performed without the
crosslinking of the sample. This is an important advantage over the
use of cross-linking agents such as formaldehyde or formaldehyde
releasers, as these reagents might reduce the recoverable amount of
extracellular nucleic acids due to cross-linking. Thus, the method
according to the present invention improves the diagnostic and
prognostic capability of the extracellular nucleic acids.
Furthermore, said stabilization allows the sample to be stored
and/or handled, e.g. transported,--even at room temperature--for a
prolonged period of time prior to separating the cells contained in
the sample and/or prior to isolating the extracellular nucleic
acids comprised therein in step b). With respect to the details of
the stabilization, it is referred to the above disclosure which
also applies here.
[0139] According to one embodiment, the cell-containing biological
sample such as e.g. a whole blood sample is stabilized in step a)
as is described in detail above using at least one apoptosis
inhibitor, at least one hypertonic agent and/or at least one
compound according to formula 1 as described above, preferably
using at least two of these stabilizing agents and optionally,
further additives. Suitable and preferred embodiments were
described above. Particularly preferred is the use of a
caspaseinhibitor in combination with an anticoagulant, preferably a
chelating agent as described above, for stabilizing whole blood
samples.
[0140] If the sample comprises large amounts of cells as is e.g.
the case with whole blood, the cells are separated from the
remaining sample in order to obtain a cell-free, respectively
cell-reduced or cell-depleted fraction of the sample which
comprises the extracellular nucleic acids. Thus, according to one
embodiment, cells are removed from the cell-containing sample
between step a) and step b). This intermediate step is only
optional and e.g. may be obsolete if samples are processed which
merely comprise minor amounts of residual cells such as e.g. plasma
or serum. However, in order improve the results it is preferred
that also respective remaining cells (or potentially remaining
cells) are removed as they might contaminate the extracellular
nucleic acid population during isolation. Depending on the sample
type, cells, including residual cells, can be separated and removed
e.g. by centrifugation, preferably high speed centrifugation, or by
using means other than centrifugation, such as e.g. filtration,
sedimentation or binding to surfaces on (optionally magnetic)
particles if a centrifugation step is to be avoided. Respective
cell removal steps can also be easily included into an automated
sample preparation protocol. Respectively removed cells may also be
processed further. The cells can e.g. be stored and/or biomolecules
such as e.g. nucleic acids or proteins can be isolated from the
removed cells.
[0141] Furthermore, it is also within the scope of the present
invention to include further intermediate steps to work up the
sample.
[0142] Extracellular nucleic acids are then isolated in step b),
e.g. from the cell-free, respectively cell-depleted fraction, e.g.
from supernatants, plasma and/or serum. For isolating extracellular
nucleic acids, any known nucleic acid isolation method can be used
that is suitable for isolating nucleic acids from the respective
sample, respectively the cell-depleted sample. Examples for
respective purification methods include but are not limited to
extraction, solid-phase extraction, silica-based purification,
magnetic particle-based purification, phenol-chloroform extraction,
chromatography, anion-exchange chromatography (using anion-exchange
surfaces), electrophoresis, filtration, precipitation, chromatin
immunoprecipitation and combinations thereof. It is also within the
scope of the present invention to specifically isolate specific
target extracellular nucleic acids, e.g. by using appropriate
probes that enable a sequence specific binding and are coupled to a
solid support. Also any other nucleic acid isolating technique
known by the skilled person can be used. According to one
embodiment, the nucleic acids are isolated using a chaotropic agent
and/or alcohol. Preferably, the nucleic acids are isolated by
binding them to a solid phase, preferably a solid phase comprising
silica or anion exchange functional groups. Suitable methods and
kits are also commercially available such as the QIAamp.RTM.
Circulating Nucleic Acid Kit (QIAGEN), the Chemagic Circulating NA
Kit (Chemagen), the NucleoSpin Plasma XS Kit (Macherey-Nagel), the
Plasma/Serum Circulating DNA Purification Kit (Norgen Biotek), the
Plasma/Serum Circulating RNA Purification Kit (Norgen Biotek), the
High Pure Viral Nucleic Acid Large Volume Kit (Roche) and other
commercially available kits suitable for extracting and purifying
circulating nucleic acids.
[0143] According to one embodiment, all nucleic acids that are
comprised in the sample that is obtained after step a) or
optionally obtained after the cells have been removed in the
intermediate step are isolated, e.g. are isolated from the
cell-free, respectively cell-depleted fraction. E.g. total nucleic
acids can be isolated from plasma or serum and the extracellular
nucleic acids will be comprised as a portion in these extracted
nucleic acids. If the cells are efficiently removed, the total
nucleic acids isolated will predominantly comprise or even consist
of extracellular nucleic acids. It is also within the scope of the
present invention to isolate at least predominantly a specific
target nucleic acid. A target nucleic acid can be e.g. a certain
type of nucleic acid, e.g. RNA or DNA, including mRNA, microRNA,
other non-coding nucleic acids, epigenetically modified nucleic
acids, and other nucleic acids. It is also within the scope of the
present invention to e.g. digest the non-target nucleic acid using
nucleases after isolation. The term target nucleic acid also refers
to a specific kind of nucleic acid, e.g. a specific extracellular
nucleic acid that is known to be a certain disease marker. As
discussed above, the isolation of extracellular nucleic acids may
also comprise the specific isolation of a respective target nucleic
acid e.g. by using appropriate capture probes. The term a target
nucleic acid also refers to a nucleic acid having a certain length,
e.g. a nucleic acid having a length of 2000 nt or less, 1000 nt or
less or 500 nt or less. Isolating respective smaller target nucleic
acids can be advantageous because it is known that extracellular
nucleic acids usually have a smaller size of less than 2000 nt,
usually less than 1000 nt and often even less than 500 nt. The
sizes, respectively size ranges indicated herein refer to the chain
length. I.e. in case of DNA it refers to bp. Focusing the
isolation, respectively purification, on respective small nucleic
acids can increase the portion of extracellular nucleic acids
obtained in the isolated nucleic acids. The stabilization methods
according to the present invention allow, in particular due to the
inhibition of fragmentation of genomic, intracellular DNA, for a
more efficient separation of such high molecular weight genomic DNA
from the fragmented extracellular nucleic acid population, e.g.,
during the nucleic acid extraction procedure. As the substantial
size difference between genomic and circulating nucleic acids is
essentially preserved using the stabilization technology according
to the present invention, genomic DNA can be removed e.g. by
size-selective recovery of DNA more efficiently than without the
respective stabilization. Suitable methods to achieve a respective
selective isolation of the extracellular nucleic acid population
e.g. by depleting the high molecular weight genomic DNA are
well-known in the prior art and thus, need no further description
here. E.g. it would be sufficient to use a size-selection method
that depletes a sample of any nucleic acid larger than 1,000-10,000
nucleotides or base pairs. As the size difference between genomic
(usually larger than >10,000 bp) and extracellular nucleic acids
(usually <1000 bp) in a stabilized sample according to the
present invention is usually relatively large due to the efficient
stabilization (the difference can e.g. lie in a range of
1000-10,000 bp), known methods for selectively isolating
extracellular nucleic acid from a biological sample could be
applied. This also provides further opportunities in order to
reduce the amount of intracellular nucleic acids in the isolated
extracellular nucleic acid population. For example, the removal of
genomic DNA during the nucleic acid extraction protocol could also
supplement or even replace a separate high g-force centrifugation
of a plasma sample before starting the nucleic acid extraction in
order to remove residual cells. Genomic DNA that is released from
said residual cells is prevented from becoming massively degraded
due to the stabilization according to the present invention, and
accordingly, can be removed by size-selective isolation protocols.
This option is of particular advantage, as many clinical
laboratories do not have a centrifuge capable of performing such a
high g-force centrifugation or other means for removing in
particular trace amounts of residual cells.
[0144] The isolated nucleic acids can then be analysed and/or
further processed in a step c) using suitable assay and/or
analytical methods. E.g. they can be identified, modified,
contacted with at least one enzyme, amplified, reverse transcribed,
cloned, sequenced, contacted with a probe, be detected (their
presence or absence) and/or be quantified. Respective methods are
well-known in the prior art and are commonly applied in the
medical, diagnostic and/or prognostic field in order to analyse
extracellular nucleic acids (see also the detailed description in
the background of the present invention). Thus, after extracellular
nucleic acids were isolated, optionally as part of total nucleic
acid, total RNA and/or total DNA (see above), they can be analysed
to identify the presence, absence or severity of a disease state
including but not being limited to a multitude of neoplastic
diseases, in particular premalignancies and malignancies such as
different forms of cancers. E.g. the isolated extracellular nucleic
acids can be analysed in order to detect diagnostic and/or
prognostic markers (e.g., fetal- or tumor-derived extracellular
nucleic acids) in many fields of application, including but not
limited to non-invasive prenatal genetic testing respectively
screening, disease screening, pathogen screening, oncology, cancer
screening, early stage cancer screening, cancer therapy monitoring,
genetic testing (genotyping), infectious disease testing, injury
diagnostics, trauma diagnostics, transplantation medicine or many
other diseases and, hence, are of diagnostic and/or prognostic
relevance. According to one embodiment, the isolated extracellular
nucleic acids are analyzed to identify and/or characterize a
disease or a fetal characteristic. Thus, as discussed above, the
isolation method described herein may further comprise a step c) of
nucleic acid analysis and/or processing. Therefore, according to
one embodiment, the isolated extracellular nucleic acids are
analysed in step c) to identify, detect, screen for, monitor or
exclude a disease and/or at least one fetal characteristic. The
analysis/further processing of the nucleic acids can be performed
using any nucleic acid analysis/processing method including, but
not limited to amplification technologies, polymerase chain
reaction (PCR), isothermal amplification, reverse transcription
polymerase chain reaction (RT-PCR), quantitative real time
polymerase chain reaction (Q-PCR), digital PCR, gel
electrophoresis, capillary electrophoresis, mass spectrometry,
fluorescence detection, ultraviolet spectrometry, hybridization
assays, DNA or RNA sequencing, restriction analysis, reverse
transcription, NASBA, allele specific polymerase chain reaction,
polymerase cycling assembly (PCA), asymmetric polymerase chain
reaction, linear after the exponential polymerase chain reaction
(LATE-PCR), helicase-dependent amplification (HDA), hot-start
polymerase chain reaction, intersequence-specific polymerase chain
reaction (ISSR), inverse polymerase chain reaction, ligation
mediated polymerase chain reaction, methylation specific polymerase
chain reaction (MSP), multiplex polymerase chain reaction, nested
polymerase chain reaction, solid phase polymerase chain reaction,
or any combination thereof. Respective technologies are well-known
to the skilled person and thus, do not need further description
here.
[0145] According to one embodiment, either or both of the isolating
or analyzing steps b) and c) occurs at least one day up to 7 days
after the sample has been collected, respectively stabilized
according to the teachings of the present invention. Suitable time
periods for which the sample, in particular a blood sample,
respectively the extracellular nucleic acid population contained
therein can be stabilized using the method according to the present
invention are also described above and also apply here. According
to one embodiment, the isolation step is performed at least one
day, at least 2 days, at least 3 days, at least 4 days, at least 5
days or at least 6 days after the sample was collected and
stabilized according to the method according to the present
invention. According to one embodiment, either or both of the
isolating or analyzing steps occur without freezing the sample
and/or without the use of formaldehyde for preserving the
cell-containing biological sample. The biological sample is
stabilized after the contact with the apoptosis inhibitor, the
hypertonic agent and/or the compound according to formula 1 as
defined above, preferably in combination with a further additive
such as an anticoagulant like EDTA. An anticoagulant is preferably
used when stabilizing blood or a sample derived from blood. The
respectively stabilized samples can be handled, e.g. stored and/or
shipped at room temperature.
[0146] Furthermore, according to a third aspect of the present
invention a composition suitable for stabilizing the extracellular
nucleic acid population in a biological sample is provided,
comprising: [0147] a) at least one apoptosis inhibitor, preferably
a caspase inhibitor, and/or [0148] b) at least one hypertonic agent
which stabilizes cells comprised in the sample, preferably
dihydroxyacetone; and/or [0149] c) at least one compound according
to formula 1 as defined above; and [0150] d) optionally at least
one anticoagulant, preferably a chelating agent.
[0151] As discussed above, a respective stabilizing composition is
particularly effective in stabilizing a cell-containing biological
sample, in particular whole blood, plasma and/or serum by
stabilizing the comprised cells and the comprised extracellular
nucleic acids thereby substantially preserving, respectively
stabilizing the extracellular nucleic acid population. A respective
stabilizing composition allows the storage and/or handling, e.g.
shipping, of the sample, which preferably is whole blood, at room
temperature for at least two, preferably at least three days
without substantially compromising the quality of the sample,
respectively the extracellular nucleic acid population contained
therein. Of course, it is not mandatory to make use of the full
possible stabilization period; the samples may also be processed
earlier if desired. Contacting the biological sample with the
stabilizing composition allows the sample to be stored, and or
handled, e.g. shipped, even at room temperature prior to isolating
and optionally analysing and/or processing the contained
circulating nucleic acids. Thus, the time between the collection or
stabilization of the sample and the nucleic acid extraction can
vary without substantially affecting the population, respectively
the composition of the extracellular nucleic acid population
contained therein. In particular, dilutions, respectively
contaminations with intracellular nucleic acids, in particular
fragmented genomic DNA, are reduced. Preferably, the stabilization
composition is contacted with the sample immediately after or
during collection of the sample. Preferably, when stabilizing a
blood sample, the composition comprises at least one caspase
inhibitor and at least one anticoagulant, preferably a chelating
agent as described above. It may also comprise further stabilizing
agents as described herein.
[0152] Suitable and preferred embodiments of the apoptosis
inhibitor, the hypertonic agent and/or the compound according to
formula 1 as well as suitable and preferred concentrations of the
respective compounds are described in detail above in conjunction
with the stabilization method. It is referred to the above
disclosure which also applies with respect to the stabilization
composition. Preferably, at least one caspase inhibitor, preferably
a modified caspase specific peptide, preferably modified at the
C-terminus with an O-phenoxy group such as Q-VD-OPh, is used in
combination with at least one hypertonic agent, preferably a
hydroxylated organic compound such as dihydroxyacetone. Other
suitable hydroxylated organic compounds are also described above,
it is referred to the respective disclosure. As is demonstrated by
the examples, a respective combination is remarkably effective in
stabilizing a cell-containing biological sample, in particular a
blood sample.
[0153] Preferably, the at least one compound according to formula 1
is a N,N-dialkyl-carboxylic acid amide. Preferred R1, R2, R3 and R4
groups are described above. According to one embodiment, the
compound is selected from the group consisting of
N,N-dimethylacetamide; N,N-diethylacetamide; N,N-dimethylformamide,
N,N-diethylformamide and N,N-dimethylpropanamid. Said compound can
also be used in combination with an apoptosis inhibitor, preferably
a caspase inhibitor (preferred embodiments are described above, it
is referred to the above disclosure) and/or a hypertonic agent,
preferably a hydroxycarbon compound (preferred embodiments are
described above, it is referred to the above disclosure).
[0154] Furthermore, it is preferred that the stabilization
composition comprises further additives, e.g. an anticoagulant such
as a chelating agent in particular if the composition is used for
stabilizing whole blood, plasma or serum.
[0155] According to one embodiment, the stabilizing composition
consists essentially of the mentioned stabilizers and optional
additives and optionally, buffering agents. The stabilizing
composition stabilizes the sample and thus, does not promote the
lysis and/or disruption of the cells contained in the sample. The
stabilizing composition may reduce the damage of the cells
comprised in the sample as can be e.g. determined by the assay
methods described in the example section.
[0156] The composition may be provided in a solid form. This is
e.g. a suitable option if the biological sample to be stabilized
contains liquid to dissolve the solid (such as for example
cell-containing body fluids, cells in medium, urine) or if liquid,
e.g. water is added thereto to dissolve the solid. The advantage of
using a solid stabilizing composition is that solids are usually
chemically more stable. However, also a liquid composition may be
used. Liquid compositions often have the advantage that the mixture
with the sample to be stabilised can be quickly achieved, thereby
basically providing an immediate stabilising effect as soon as the
sample comes into contact with the liquid stabilizing composition.
Preferably, stabilising agent(s) present in the liquid stabilizing
composition remain stable in solution and require no
pre-treatment-such as for example the dissolving of precipitates of
limited solubility-by the user because pre-treatments of this kind
pose a risk of variations in the stabilising efficiency.
[0157] Also provided is a mixture comprising the stabilizing
composition according to the present invention mixed with a
biological sample. Suitable and preferred examples of biological
samples as well as suitable concentrations of the stabilizing
agent(s) when mixed with the biological sample are described above
in conjunction with the stabilizing method. It is referred to the
above disclosure which also applies here. Preferably, the
stabilizing composition is pre-filled in a sample collection device
so that the sample is immediately stabilized during collection.
According to one embodiment, the stabilizing composition is
contacted with the biological sample in a volumetric ratio selected
from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5. It is a
particular advantage of the stabilizing composition of the present
invention that stabilization of a large sample volume can be
achieved with a small volume of the stabilizing composition.
Therefore, preferably, the ratio of stabilizing composition to
sample lies in a range from 1:2 to 1:7, more preferred 1:3 to
1:5.
[0158] The stabilizing composition according to the third aspect of
the present invention can be used to stabilize the extracellular
nucleic acid population comprised in a cell-containing sample.
Furthermore, the stabilizing composition according to the third
aspect of the present invention may also be used for stabilizing
cells contained in a sample. As described above, the stabilizing
composition inter alia reduces the release of genomic DNA from
cells that results from decaying cells. Thus, a respective use is
also an advantageous and provided by the teachings according to the
present invention.
[0159] Also provided is a method of manufacturing a composition
according to the third aspect of the present invention is provided,
wherein the components of the composition are mixed, preferably in
an aqueous solution.
[0160] The composition of the present invention may also be
incorporated into a sample collection device, in particular blood
collection assembly, thereby providing for a new and useful version
of such a device. Such devices typically include a container having
an open and a closed end. The container is preferably a blood
collection tube. The container type also depends on the sample to
be collected, other suitable formats are described below.
[0161] Furthermore, the present invention provides a container for
collecting a cell-containing biological sample, preferably a blood
sample, wherein the container comprises a stabilizing composition
according to the present invention. Providing a respective
container, e.g. a sample collection tube, which comprises the
stabilizing composition according to the present invention, has the
advantage that the sample is quickly stabilized when the sample is
collected in the respective container. Details with respect to the
stabilizing composition were described above, it is referred to the
above disclosure which also applies here.
[0162] According to one embodiment, a collection container for
receiving and collecting a biological sample is provided wherein
the container comprises: [0163] a) at least one apoptosis inhibitor
such that when the sample is collected, the concentration of the
apoptosis inhibitor or combination of two or more apoptosis
inhibitors in the resulting mixture is selected from at least 0.01
.mu.M, at least 0.05 .mu.M, at least 0.1 .mu.M, at least 0.5 .mu.M,
at least 1 .mu.M, at least 2.5 .mu.M or at least 3.5 .mu.M and
preferably is present in a concentration range selected from 0.01
.mu.M to 100 .mu.M, 0.05 .mu.M to 100 .mu.M, 0.1 .mu.M to 50 .mu.M,
1 .mu.M to 40 .mu.M, 1.0 .mu.M to 30 .mu.M or 2.5 .mu.M to 25 .mu.M
and/or [0164] b) at least one hypertonic agent such that when the
sample is collected, the concentration of the hypertonic agent or
combination of two or more apoptosis inhibitors in the resulting
mixture is at least 0.05M, at least 0.1M, preferably at least
0.25M, and preferably is present in a concentration range from
0.05M to 2M, 0.1M to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M or 0.1M to
0.6M; and/or [0165] c) at least one compound according to formula 1
as defined above, such that when the sample is collected the
compound according to formula 1 is comprised in a concentration of
at least 0.1%, at least 0.5%, at least 0.75%, at least 1%, at least
1.25% or at least 1.5% or wherein said compound is comprised in a
concentration range selected from 0.1% up to 50%. 0.1 to 30%, 1% to
20%, 1% to 10%, 1% to 7.5% and 1% to 5%; and/or [0166] d)
optionally at least one further additive, preferably an
anticoagulant such as a chelating agent, preferably EDTA if the
container is for collecting blood or a blood product. Suitable
concentrations are described above and preferably lie in the range
of 4 mM to 50 mM, more preferred 4 mM to 20 mM.
[0167] The pre-filled components a), b), c) and/or d) can be
provided in a liquid or in a dry form. For stabilizing whole blood
it is preferred to use at least components a) and d). Preferably,
the stabilizing components are provided as a stabilizing
composition. A dry form is e.g. a suitable option if the biological
sample to be stabilized contains liquid to dissolve the solid (such
as for example cell-containing body fluids, cells in medium, urine)
or if liquid, e.g. water is added thereto to dissolve the solid.
The advantage of using a solid stabilizing composition is that
solids are usually chemically more stable than liquids. According
to one embodiment, the inner wall of the container is
treated/covered with a stabilizing composition according to the
present invention. Said composition can be applied to the inner
walls using e.g. a spray-dry-method. Liquid removal techniques can
be performed on the stabilising composition in order to obtain a
substantially solid state protective composition. Liquid removal
conditions may be such that they result in removal of at least
about 50% by weight, at least about 75% by weight, or at least
about 85% by weight of the original amount of the dispensed liquid
stabilising composition. Liquid removal conditions may be such that
they result in removal of sufficient liquid so that the resulting
composition is in the form of a film, gel or other substantially
solid or highly viscous layer. For example it may result in a
substantially immobile coating (preferably a coating that can be
re-dissolved or otherwise dispersed upon contact with the
cell-containing sample which preferably is a blood product sample).
It is possible that lyophilization or other techniques may be
employed for realizing a substantially solid form of the protective
agent (e.g., in the form of one or more pellet). Thus, liquid
removal conditions may be such that they result in a material that
upon contact with the sample under consideration (e.g., a whole
blood sample) the protective agent will disperse in the sample, and
substantially preserve components (e.g., extracellular nucleic
acids) in the sample. Liquid removal conditions may be such that
they result in a remaining composition that is substantially free
of crystallinity, has a viscosity that is sufficiently high that
the remaining composition is substantially immobile at ambient
temperature; or both.
[0168] However, also a liquid composition may be used. Liquid
compositions often have the advantage that the mixture with the
sample to be stabilised can be quickly achieved, thereby basically
providing an immediate stabilising effect as soon as the sample
comes into contact with the liquid stabilizing composition.
Preferably, the stabilising agent(s) present in the liquid
stabilizing composition remain stable in solution and require no
pre-treatment--such as for example the dissolving of precipitates
of limited solubility--by the user because pre-treatments of this
kind pose a risk of variations in the stabilising efficiency.
[0169] The stabilizing composition is comprised in the container in
an amount effective to provide the stabilisation of the amount of
sample to be collected in said container. According to one
embodiment, the liquid stabilizing composition is contacted with
the biological sample in a volumetric ratio selected from 10:1 to
1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5. It is a particular
advantage of the stabilizing composition of the present invention
that stabilization of a large sample volume can be achieved with a
small volume of the stabilizing composition. Therefore, preferably,
the ratio of stabilizing composition to sample lies in a range from
1:2 to 1:7, more preferred 1:3 to 1:5.
[0170] According to one embodiment, the container is evacuated. The
evacuation is preferably effective for drawing a specific volume of
a fluid sample into the interior. Thereby, it is ensured that the
correct amount of sample is contacted with the pre-filled amount of
the stabilizing composition comprised in the container, and
accordingly, that the stabilization is efficient. According to one
embodiment, the container comprises a tube having an open end
sealed by a septum. E.g. the container is pre-filled with a defined
amount of the stabilizing composition either in solid or liquid
form and is provided with a defined vacuum and sealed with a
septum. The septum is constructed such that it is compatible with
the standard sampling accessories (e.g. cannula, etc.). When
contacted with e.g. the canula, a sample amount that is
predetermined by the vacuum is collected in the container. A
respective embodiment is in particular advantageous for collecting
blood. A suitable container is e.g. disclosed in U.S. Pat. No.
6,776,959.
[0171] The container according to the present invention can be made
of glass, plastic or other suitable materials. Plastic materials
can be oxygen impermeable materials or may contain an oxygen
impermeable layer. Alternatively, the container can be made of
water- and air-permeable plastic material. The container according
to the present invention preferably is made of a transparent
material. Examples of suitable transparent thermoplastic materials
include polycarbonates, polyethylene, polypropylene and
polyethyleneterephthalate. The container may have a suitable
dimension selected according to the required volume of the
biological sample being collected. As described above, preferably,
the container is evacuated to an internal pressure below
atmospheric pressure. Such an embodiment is particularly suitable
for collecting body fluids such as whole blood. The pressure is
preferably selected to draw a predetermined volume of a biological
sample into the container. In addition to such vacuum tubes also
non-vacuum tubes, mechanical separator tubes or gel-barrier tubes
can be used as sample containers, in particular for the collection
of blood samples. Examples of suitable containers and capping
devices are disclosed in U.S. Pat. No. 5,860,397 and US
2004/0043505. As container for collecting the cell-containing
sample also further collection devices, for example a syringe, a
urine collection device or other collection devices can be used.
The type of the container may also depend on the sample type to be
collected and suitable containers are also available to the skilled
person.
[0172] In an advantageous embodiment the container respectively the
device is filled or is pre-filled with at least one apoptosis
inhibitor, preferably a caspase inhibitor, at least one hypertonic
agent, preferably at least one hydroxylated organic compound as
described in detail above, e.g. dihydroxyaceton and optionally a
further additive such as an anticoagulant, preferably a chelating
agent, more preferred EDTA. The mixture of at least one hypertonic
agent, which preferably is a hydroxylated organic compound, e.g. a
carbohydrate such as dihydroxyacetone and at least one caspase
inhibitor, preferably Q-VD-OPH, unexpectedly stabilizes
extracellular nucleic acids in whole blood, plasma or serum and
prevents the release of cellular nucleic acids in particular from
white blood cells that are contained in such samples. Hence, the
extracellular nucleic acid population is preserved in the state it
had shown at the time of blood draw. Beneficial results are also
obtained when the container respectively the device is filled or is
pre-filled with at least one compound according to formula 1 as
defined above as stabilizing agent. Preferably, an anticoagulant is
encompassed in addition to the compound according to formula 1. The
anticoagulant is preferably a chelating agent such as EDTA.
Furthermore, the stabilizing composition comprised in the container
may also comprise an apoptosis inhibitor, preferably a caspase
inhibitor and/or at least one hypertonic agent, preferably at least
one hydroxylated organic compound as described in detail above,
e.g. dihydroxyaceton and optionally further additives. According to
one embodiment, the stabilizing composition comprised in the
container comprises a caspase inhibitor and an anticoagulant.
[0173] According to one embodiment, the container has an open top,
a bottom, and a sidewall extending therebetween defining a chamber,
wherein the stabilization composition according to the present
invention is comprised in the chamber. It may be comprised therein
in liquid or solid form. According to one embodiment the container
is a tube, the bottom is a closed bottom, the container further
comprises a closure in the open top, and the chamber is at a
reduced pressure. The advantages of a reduced pressure in the
chamber were described above. Preferably, the closure is capable of
being pierced with a needle or cannula, and the reduced pressure is
selected to draw a specified volume of a liquid sample into the
chamber. According to one embodiment, the chamber is at a reduced
pressure selected to draw a specified volume of a liquid sample
into the chamber, and the stabilizing composition is a liquid and
is disposed in the chamber such that the volumetric ratio of the
stabilising composition to the specified volume of the
cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15,
1:1 to 1:10 and 1:2 to 1:5. The associated advantages were
described above.
[0174] Preferably, the container is for drawing blood from a
patient.
[0175] According to a fifth aspect, a method is provided comprising
the step of collecting a sample from a patient directly into a
chamber of a container according to the fourth aspect of the
present invention. Details with respect to the container and the
sample were described above. It is referred to the respective
disclosure. According to one embodiment, a blood sample is
collected, preferably it is withdrawn from the patient.
[0176] The methods and compositions disclosed herein allow for the
efficient preservation and isolation of extracellular nucleic acids
while reducing possible mixing with nucleic acids, in particular
fragmented genomic DNA, which originates from cells comprised in
the biological sample and which may enter a biological sample due
to cell damage, respectively cell lysis. The methods according to
the present invention, as well as the compositions and the
disclosed devices (e.g. the collection containers) reduce the
degradation of extracellular nucleic acids and also reduce cell
lysis and/or release of genomic nucleic acids, in particular
fragmented genomic DNA, so that the extracellular nucleic acids
contained in the sample do not become contaminated with
intracellular nucleic acids, respectively a respective
contamination is reduced by the teachings according to the present
invention. As discussed above, an intermixing of extracellular
nucleic acids and cellular nucleic acids, in particular fragmented
genomic DNA, may reduce the accuracy of any measurement of the
amount of extracellular nucleic acids in a biological sample. As
discussed above, an important advantage of the present invention is
the possibility for essentially simultaneous stabilizing of both
the cells contained in the sample (in particular white blood cells
in case of whole blood, plasma or serum) and the extracellular
nucleic acids. This helps to prevent cellular nucleic acids such as
genomic DNA from being released into the cell-free portion of the
sample, and further diluting the comprised extracellular nucleic
acids (and associated biomarkers) of interest, while also
maintaining the structural integrity of the extracellular nucleic
acids. As discussed herein, contacting the cell-containing
biological sample such as whole blood or plasma with the
stabilising agent(s) allows the sample to be stored for a period of
time prior to isolating the extracellular nucleic acids. More
preferably, the cell-containing biological sample, e.g. blood or
plasma, may be drawn at one location (e.g., a health care
facility), contacted with the stabilising agent(s), and later
transported to a different remote location (e.g., a laboratory) for
the nucleic acid isolation and testing process.
[0177] Furthermore, the stabilization reagents, as disclosed in
herein, provide an advantage over known state-of-the-art
stabilization reagents which involve the use of cross-linking
reagents, such as formaldehyde, formaldehyde releasers and the
like, as the stabilization of samples according to the present
invention does not involve the use to such crosslinking reagents.
Crosslinking reagents cause inter- or intra-molecular covalent
bonds between nucleic acid molecules or between nucleic acids and
proteins. This effect can lead to a reduced recovery of such
stabilized and partially crosslinked nucleic acids after a
purification or extraction from a complex biological sample. As,
for example, the concentration of circulating nucleic acids in a
whole blood samples is already relatively low, any measure which
further reduces the yield of such nucleic acids should be avoided.
This may be of particular importance when detecting and analyzing
very rare nucleic acid molecules derived from malignant tumors or
from a developing fetus in the first trimester of pregnancy.
Therefore, according to one embodiment, no formaldehyde releaser is
comprised in the stabilizing composition, respectively is not
additionally used for stabilization. According to one embodiment,
the apoptosis inhibitor that is used in the methods and/or
compositions according to the present invention is not selected
from the group consisting of aurintricarboxylic acid,
phenylmethylsulfonyl fluoride (PMSF), leupeptin and Na-Tosyl-Lys
chloromethyl ketone hydrochloride (TLCK). According to one
embodiment, the apoptosis inhibitor is not selected from said group
in particular if the apoptosis inhibitor is not used in combination
with a hypertonic agent as additional stabilizer.
[0178] This invention is not limited by the exemplary methods and
materials disclosed herein, and any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of embodiments of this invention. Numeric ranges are
inclusive of the numbers defining the range. The headings provided
herein are not limitations of the various aspects or embodiments of
this invention which can be read by reference to the specification
as a whole.
[0179] The term "solution" as used herein in particular refers to a
liquid composition, preferably an aqueous composition. It may be a
homogenous mixture of only one phase but it is also within the
scope of the present invention that a solution comprises solid
additives such as e.g. precipitates.
[0180] The sizes, respectively size ranges indicated herein with
reference to nucleotides nt, refer to the chain length and thus are
used in order to describe the length of single-stranded as well as
double-stranded molecules. In double-stranded molecules said
nucleotides are paired.
[0181] According to one embodiment, subject matter described herein
as comprising certain steps in the case of methods or as comprising
certain ingredients in the case of compositions, solutions and/or
buffers refers to subject matter consisting of the respective steps
or ingredients. It is preferred to select and combine preferred
embodiments described herein and the specific subject-matter
arising from a respective combination of preferred embodiments also
belongs to the present disclosure.
TABLE-US-00002 TABLE 1 Overview of apoptosis inhibitors Apoptosis
inhibitor Description 1. Metabolic inhibitors AICA-Riboside,
Acadesine, Offers protection against cell death induced by glucose
deprivation AICAr, 5-Aminoimidazole-4-
carboxamide-1-.beta.-riboside, Z- Riboside Apoptosis Inhibitor II,
diarylurea prevents the active ~700-kDa apoptosome complex
formation compound Bax Channel Blocker, (.+-.)-1- A cell-permeable
dibromocarbazolo-piperazinyl derivative that
(3,6-Dibromocarbazol-9-yl)-3- displays anti-apoptotic properties.
Effectively blocks Bid-induced piperazin-1-yl-propan-2-ol, bis
cytochrome c release from HeLa cell mitochondria (~80% TFA, iMAC1
inhibition at 5 .mu.M) by inhibiting Bax channel-forming activity
(IC50 = 520 nM in a liposome channel assay). Bax-Inhibiting
Peptide, V5 A cell-permeable pentapeptide based on the Ku70-Bax
inhibiting domain that offers cytoprotection. Functions as
effectively as the Peptide sequence: Caspase Inhibitor VI
(Z-VAD-FMK; Cat. No. 219007) for Bax- H-Val-Pro-Met-Leu-Lys-OH
mediated apoptosis (~50-200 .mu.M). Also effectively blocks
caspase- independent necrotic cell death. Shown to be Ku70
competitive, interact with Bax, prevent its conformational change
and mirochondrial translocation. Displays extended stability in
culture medium (~3 days). Negative control peptide is also
available Bcl-xL BH44-23, Human, Cell- A cell-permeable peptide
that prevents apoptotic cell death by Permeable directly binding to
the voltage-dependent anion channel (VDAC) and blocking its
activity. Leads to the inhibition of cytochrome c release and loss
of mitochondrial membrane potential (.DELTA..psi.m). Contains the
conserved N-terminal homology domain (BH4) of Bcl- xL (amino acids
4-23) that has been shown to be essential for inhibiting VDAC
activity in liposomes and in isolated mitochondria. The BH4 domain
is linked to a carrier peptide, a 10-amino acid HIV-TAT48-57
sequence with a .beta.-alanine residue as a spacer for maximum
flexibility. Following its uptake, it is mainly localized to the
mitochondria Bongkrekic Acid, Triammonium Acts as a ligand of the
adenine nucleotide translocator. A potent Salt inhibitor of
mitochondrial megachannel (permeability transition pore).
Significantly reduces signs of apoptodsis induced by nitric oxide.
Prevents the apoptotic breakdown of the inner mitochondiral
transmembrane potential (.DELTA..psi.m), as well as a number of
other phenomena linked to apoptosis Duanorubicin, Hydrochloride
Potent cell-permeable anticancer agent whose potential target site
may be mitochondiral cytochrome c oxidase. Has been shown to
inhibit RNA and DNA synthesis. Inhibits eukaryotic topoisomerases I
and II. Induces DNA single-strand breaks. Also induces apoptosis in
HeLa S3 tumor cells. According to one embodiment, said compound is
not used as stabilizer according to the present invention. Humanin,
Human, Synthetic A 24-residue anti-apoptotic peptide that, when
expressed intracellularly, offers protection against neuronal
apoptosis induced by presenilin and APP (amyloid precursor protein)
mutants associated with familial Alzheimer's disease (AD). Shown to
reduce cytochrome c release in vitro by directly binding to Bax
(Bcl-2-associated X protein; Kd ~ 2 nM) and preventing its
association with isolated mitochondria
Phorbol-12-myristate-13-acetate Most commonly-used phorbol ester.
Extremely potent mouse skin tumor promoter. Activates protein
kinase C in vivo and in vitro, even at nM concentrations. Promotes
the expression of inducible NOS in cultured hepatocytes. Activates
Ca2+-ATPase and potentiates forskolin-induced cAMP formation.
Inhibits apoptosis induced by the Fas antigen, but induces
apoptosis in HL-60 promyelocytic leukemia cells. Its binding is
reversible Pifithrin-.alpha. A cell-permeable chemical inhibitor of
p53. Reversibly inhibits p53- dependent transactivation of
p53-responsive genes and reversibly blocks p53-mediated apoptosis.
Inhibits p53-dependent growth arrest of human diploid fibroblasts
in response to DNA damage but has no effect on p53-deficient
fibroblasts. Protects normal tissues from the deleterious side
effects of chemotherapy. Has been reported to protect neurons
against .beta.-amyloid peptide and glutamate-induced apoptosis
Pifithrin-.mu. A cell-permeable sulfonamide that blocks p53
interaction with Bcl- xL and Bcl-2 proteins and selectively
inhibits p53 translocation to mitochondria without affecting the
transactivation function of p53. Effectively protects against
.gamma. radiation-induced cell death in vitro and animal lethality
in vivo. Because Pifithrin-p targets only the mitochondrial branch
of the p53 pathway without affecting the important transcriptional
functions of p53, it is superior to Pifithrin-a (Cat. No. 506132)
in in vivo studies. Shown to selectively interact with inducible
HSP70 and disrupt its functions Pifithrin-.alpha., Cyclic- A
cell-permeable and very stable analog of Pifithrin-a (Cat. No.
506132), with similar biological function, but with reduced
cytotoxicity. A chemical inhibitor of p53. Reversibly inhibits p53-
dependent transactivation of p53-responsive genes; also reversibly
blocks p53-mediated apoptosis. Acts as a P-gp modulator by changing
relative substrate specificity of the transporter. This compound
has been reported to be a potent STAT6 transcriptional inhibitor
Pifithrin-.alpha., p-Nitro A cell-permeable p53 inhibitor that
serves as the prodrug form of Pifithrin-.alpha., p-Nitro, Cyclic
(Cat. No. 506154). Although its in vitro efficacy (ED50 = 0.3 .mu.M
in protecting etoposide-induced cortical neuron death) is similar
to that of Pifithrin-.alpha. (Cat. No. 506132), it is 100-fold more
potent than Pifithrin-.alpha.when adminstered in rats in vivo due
to its long-lasting, steady conversion to the corresponding cyclic
form of active compound in biological systems (t1/2 = 8 h in neuron
culture medium at 37.degree. C.). Pifithrin-.alpha., p-Nitro,
Cyclic A cell-permeable p53 inhibitor that exhibits 10-fold higher
potency (ED50 = 30 nM in protecting etoposide-induced cortical
neuron death) and 50% longer half-life (t1/2 = 6 h in neuron
culture medium at 37.degree. C.) than Pifithrin-.alpha. (Cat. No.
506132). However, despite its in vitro efficacy, this inhibitor is
not effective when adminstered in rats in vivo. For in vivo
applications, please consider Pifithrin-.alpha., p-Nitro (Cat. No.
506152). STAT3 Inhibitor Peptide A Stat3-SH2 domain binding
phosphopeptide that acts as a selective inhibitor of Stat3 (signal
transducers and activators of Peptide sequence: transcription 3)
signaling with a DB50 of 235 .mu.M (concentration of
Ac-Pro-Tyr(PO3H2)-Leu-Lys- peptide at which DNA-binding activity is
inhibited by 50%). Thr-Lys-OH Significantly lowers the DNA-binding
activity of Stat3 by forming an inactive Stat3:peptide complex and
reduces the levels of active Stat3:Stat3 dimers that can bind DNA.
Displays greater affinity for Stat3, and to a lesser extent Stat1,
over Stat5. Supplied as a trifluoroacetate salt. STAT3 Inhibitor
Peptide, Cell- A cell-permeable analog of the Stat3-SH2
domain-binding Permeable phosphopeptide (Cat. No. 573095) that
contains a C-terminal mts (membrane translocating sequence) and
acts as a highly selective, Peptide sequence: potent blocker of
Stat3 activation. Also suppresses constitutive
Ac-Pro-Tyr(PO3H2)-Leu-Lys- Stat-3 dependent Src transformation with
no effect on Stat-3 Thr-Lys-OH independent Ras transformation. The
unphosphorylated inactive control peptide is also available under
Cat. No. 573105. Supplied as a trifluoroacetate salt. CAY10500,
6,7-dimethyl-3- Tumor necrosis factor a (TNF.alpha.) inhibitor that
prevents binding to {[methyl-[1-(3-trifluoromethyl- the TNF
Receptor 1 (TNFR1).6 Binds to the biologically active
phenyl)-1H-indol-3-ylmethyl]- TNF.alpha. trimer and promotes
accelerated displacement of a single amino}-ethyl)-amino]-methyl}-
subunit to rapidly inactivate the cytokine. In a cell based assay,
chromen-4-one compound inhibited TNF.alpha.-mediated stimulation of
IKB degradation. Gambogic amide A selective agonist for TrkA which
mimics the actions of NGF. This compound possesses robust
neurotrophic actvity, while it prevents neuronal cell death 1.
Maslinic Acid A pentacyclic triterpene with antioxidant and
anti-inflammatory properties. Shown to block the generation of
nitric oxide, and inhibits the secretion of IL-6 and TNF-a induced
by lipopolysaccharides Naringin hydrate A citrus bioflavonoid found
to inhibit cytochrome P450 monooxygenase activity in mouse liver.
It prevents toxin-induced cytoskeletal disruption and apoptotic
liver cell death. Necrostatin-1 An inhibitor of necroptosis, a
non-apoptotic cell death pathway. Does not affect
Fas/TNFR-triggered apoptosis. According to one embodiment, said
compound is not used as stabilizer according to the present
invention. NSC348884 hydrate, N1,N2- This product is a nucleolar
phosphoprotein that displays several
bis((3-imino-6-methyl-3H-indol- biological activities in ribosome
biogenesis, cell proliferation, 2-yl)methyl)-N1,N2-bis((6-
cytoplasmic/nuclear shuttle transportation, nucleic acid binding,
methyl-1H-benzo[d]imidazol-2- ribonucleic cleavage, centrosome
duplication and molecular yl)methyl)ethane-1,2-diamine chaperoning,
and is found in higher levels in tumor cells. hydrate
Overexpression has been shown to lead to inhibition of apoptosis.
NSC34884 upregulates p53. Orsellinic acid Benzoic acid. Blocks
PAF-mediated neuronal apoptosis. Shows free radical scavenging
activity. tetramethyl A synthetic derivative of NDGA and a
non-selective lipoxygenase Nordihydroguaiaretic Acid inhibitor. It
inhibits Sp1 transcription factor binding at the HIV long terminal
repeat promoter and at the .alpha.-ICP4 promoter (a gene essential
for HSV replication). GW 4869, 3,3'-(1,4- A cell-permeable,
symmetrical dihydroimidazolo-amide compound
phenylene)bis[N-[4-(4,5- that acts as a potent, specific,
non-competitive inhibitor of N- dihydro-1H-im idazol-2- SMase
(neutral sphingomyelinase) [IC50 = ~ 1 .mu.M, rat brain; Km
yl)phenyl]-hydrochloride-2- for sphingomyelin ~13 .mu.M]. Does not
inhibit human A-SMase (acid propenamide sphingomyelinase) even at
150 .mu.M. Weakly inhibits the activities of bovine protein
phosphatase 2A and mammalian lyso-PAF PLC, while no inhibition is
observed for bacterial phosphatidylcholine- specific PLC. Reported
to offer complete protection against TNF-.alpha. or diamine-induced
cell death in MCF7 breast cancer cells at 20 .mu.M. Does not modify
the intracellular glutathione levels or interfere with TNF-.alpha.
or diamine-mediated signaling effects. SP 600125, 1,9- SP600125 is
a JNK inhibitor (IC50 = 40 nM for JNK-1 and JNK-2 Pyrazoloanthrone,
and 90 nM for JNK-3). This agent exhibits greater than 300-fold
Anthrapyrazolone selectivity for JNK against related MAP kinases
ERK1 and p38-2, and the serine threonine kinase PKA. [1] SP600125
is a reversible ATP-competitive inhibitor. In cells, SP600125 dose
dependently inhibited the phosphorylation of c-Jun, the expression
of inflammatory genes COX-2, IL-2, IFN-y, TNF-.alpha., and
prevented the activation and differentiation of primary human CD4
cell cultures Mdivi-1, 3-(2,4-Dichloro-5- Mdivi-1 is a selective
inhibitor of mitochondrial division in yeast and
methoxyphenyl)-2,3-dihydro-2- mammalian cells which acts via
inhibiting the mitochondrial division thioxo-4(1H)-quinazolinone,
3- dynamin. In cells, Mdivi-1 inhibits apoptosis by inhibiting
(2,4-Dichloro-5-methoxyphenyl)- mitochondrial outer membrane
permeabilization. Mdivi-1 causes 2-sulfanyl-4(3H)-quinazolinone the
rapid (<5 min) reversible and dose-dependent formation of net-
like mitochondria in wild-type cells with an IC50 = ~10 .mu.M. In
yeast, time-lapse fluorescence microscopy revealed no detectable
mitochondrial division after treatment with Mdivi-1 Minocycline .
hydrochloride Tetracycline derivative with antimicrobial activity.
Inhibitor of angiogenesis, apoptosis and poly(ADP-ribose)
polymerase-1 (PARP-1). Anti-inflammatory and neuroprotective Ro
08-2750 (C13H10N4O3) Inhibitor of NGF-induced apoptosis. RKTS-33
(C7H8O4) selective inhibition of Fas ligand-dependent pathway alone
2. Nucleic acids 3,4-Dichloroisocoumarin Inhibitor of serine
proteases .fwdarw. granzyme B and blocks apoptotic internucleosomal
DNA cleavage in thymocytes without the
involvement of endonucleases. Does not affect thiol proteases and
metalloproteases Actinomycin D, Streptomyces Also acts as a
competitive inhibitor of serine proteases; Classical sp.
anti-neoplastic drug. Cytotoxic inducer of apoptosis against tumor
cells. A DNA dependent inhibitor of RNA synthesis, actinomycin
promotes induction of apoptosis by some specific stimuli, for
example, TRAIL and Fas (CD95). Actinomycin D can also alleviate or
block the apoptotic process and decrease the cytotoxicity induced
by several stimuli such as the dihydrofolate reductase inhibitor
aminopterin and the prostaglandin derivative
15-deoxy-D12,14-prostaglandin J2, thus it can have both pro and
anti-apoptotic activities in some systems. According to one
embodiment, said compound is not used as stabilizer according to
the present invention. Aurintricarboxylic Acid Inhibitor of DNA
topoisomerase II Baicalein A cell-permeable flavone that inhibits
the activity of 12- lipoxygenase (IC50 = 120 nM) and reverse
transcriptase. Protects cortical neurons from .beta.-amyloid
induced toxicity. Reduces leukotriene biosynthesis and inhibits the
release of lysosomal enzymes. Also inhibits cellular Ca2+ uptake
and mobilization, and adjuvant-induced arthritis. Reported to
inhibit microsomal lipid peroxidation by forming an iron-baicalein
complex. Inhibits topoisomerase II and induces cell death in
hepatocellular carcinoma cell lines. Potentiates contractile
responses to nerve stimulation. Inhibits protein tyrosine kinase
and PMA-stimulated protein kinase C Camptothecin, Camptotheca A
cell-permeable DNA topoisomerase I inhibitor. Exhibits anti-
acuminata leukemic and antitumor properties. Induces apoptosis in
HL-60 cells and mouse thymocytes. Arrests cells at the G2/M phase
Diisopropylfluorophosphate serine protease inhibitor
Phenylmethylsulfonyl Fluoride Irreversible inhibitor of serine
proteases. Its mechanism of action is (PMSF) analogous to that of
diisopropylfluorophosphate. PMSF causes sulfonylation of the
active-site serine residues. Also reported to inhibit
internucleosomal DNA fragmentation in immature thymocytes. For a
related, more stable inhibitor, see AEBSF (-)-Huperzine A An
inhibitor of AChE. Antagonist of NMDA receptors. Protects against
glutamate-mediated excitotoxicity. Razoxane Inhibits topoisomerase
II without inducing DNA strand breaks (topo II catalytic
inhibitor). Suptopin-2 Suppressor of topoisomerase II inhibition.
Reverses cell cycle arrest; bypass of checkpoint function. Has
inherent fluorescence and a distinct advantage in identification of
molecule targets; effective concentraion in the pM range. 3.
Enzymes 3.1. Caspases Apoptosis Inhibitor; 2-(p- Effects
attributable to the inhibition of caspase-3 activation
Methoxybenzyl)-3,4- pyrrolid inedio1-3-acetate cIAP-1, Human,
Recombinant, Recombinant, human cIAP-1 (amino acids 1-618) fused to
the E. coli peptide sequence MATVIDH10SSNG at the N-terminus and
expressed in E. coli. clAP is a member of the inhibitor of
apoptosis family of proteins that inhibits proteolytic activity of
mature caspases by interaction of the BIR domain with the active
caspase CrmA, Recombinant CrmA (cowpox viral serpin cytokine
response modifier A) is purified from E. coli transformed with a
construct containing the full-length coding region of the CrmA gene
and 7 additional amino acids that do not affect the activity. CrmA
is a natural inhibitor of human caspase-1 and granzyme B, enzymes
that are involved in apoptosis Group III Caspase Inhibitor I A
potent, cell-permeable, and irreversible inhibitor of Group III
caspases (caspase-6, -8, -9, and -10), although more effective
Peptide sequence: towards caspases-6 and -8. Also inhibits
caspase-1 and caspase- Ac-I le-Glu-Pro-Asp-CHO, Ac- 3. When using
with purified native or recombinant enzyme, IEPD-CHO, Caspase-8
inhibitor pretreatment with an esterase is required. III Kaempferol
A cell-permeable phytoestrogen that inhibits topoisomerase I-
catalyzed DNA religation in HL-60 cells. Offers protection against
A.beta.5-35-induced cell death in neonatal cortical neurons. Its
protective effects are comparable to that of estradiol. Blocks the
A.beta.-induced activation of caspase-2, -3, -8, and -9, and
reduces NMDA-induced neuronal apoptosis. Reported to be a potent
inhibitor of monoamine oxidases. Acts as an inhibitor of COX-1
activity (IC50 = 180 .mu.M), and of transcriptional activation of
COX-2 (IC50<15 .mu.M Q-VD-OPH General, Pancaspase Boc-D(OMe)-FMK
General, Pancaspase Z-D(OMe)E(OMe)VD(OMe)- Caspase 3, 7 FMK
Z-LE(OMe)TD(OMe)-FMK Caspase 8 Z-YVAD(OMe)-FMK Caspase 1, 4
Z-FA-FMK Inhibits Cathepsin B Z-FF-FMK Cathepsin B, L
Mu-PheHphe-FMK Cathepsin B, L Z-AE(OMe)VD(OMe)-FMK Caspase 10
Z-ATAD(OMe)-FMK Caspase 12 Z-VK(Biotin)-D(OMe)-FMK General Caspase
Z-LE(OMe)VD(OMe)-FMK Caspase 4 Z-VAM-FMK Antiviral peptide
inhibitor, Inhibits HRV2 and HRV14 4'-Azidocytidine HCV Inhibitor
Caspase-13 Inhibitor I A potent, reversible inhibitor of caspase-13
(ERICE). Peptide sequence: Ac-Leu-Glu-Glu-Asp-CHO Caspase-13
Inhibitor II A cell-permeable, irreversible inhibitor of
caspase-13. When using with purified native or recombinant enzyme,
pretreatment with an Peptide sequence: esterase is required.
Z-Leu-Glu(OMe)-Glu(OMe)- Asp(OMe)-FMK Caspase-1 Inhibitor I A
potent, specific, and reversible inhibitor of caspase-1 (Ki = 200
pM for human recombinant caspase-1), caspase-4, and caspase-
Peptide sequence: 5. Strongly inhibits anti-APO-1 induced apoptosis
in L929-APO-1 Ac-Tyr-Val-Ala-Asp-CHO cells. Caspase-1 Inhibitor I,
Cell- A cell-permeable inhibitor of caspase-1 (ICE;
Interleukin-1.beta. Permeable Converting Enzyme), caspase-4, and
caspase-5. The C-terminal YVAD-CHO sequence of this peptide is a
highly specific, potent, Peptide sequence: and reversible inhibitor
of caspase-1 (Ki = 1 nM). The N-terminal
Ac-Ala-Ala-Val-Ala-Leu-Leu- sequence (amino acid residues 1-16)
corresponds to the Pro-Ala-Val-Leu-Leu-Ala-Leu- hydrophobic region
(h-region) of the signal peptide of the Kaposi
Leu-Ala-Pro-Tyr-Val-Ala-Asp- fibroblast growth factor (K-FGF) and
confers cell-permeability to CHO the peptide Caspase-1 Inhibitor II
A cell-permeable and irreversible inhibitor of caspase-1 (Ki = 760
pM), caspase-4, and caspase-5. Inhibits Fas-mediated apoptosis
Peptide sequence: and acidic sphingomyelinase activation
Ac-Tyr-Val-Ala-Asp-CM K Caspase-1 Inhibitor IV A highly selective,
competitive, cell-permeable, and irreversible inhibitor of
caspase-1, caspase-4, and caspase-5. Inactivates the Peptide
sequence: enzyme with a rate limited by diffusion and is relatively
inert toward Ac-Tyr-Val-Ala-Asp-AOM (AOM = other bionucleophiles
such as glutathione, making it an excellent
2,6-dimethylbenzoyloxymethyl candidate for in vivo studies of
enzyme inhibition ketone) Caspase-1 Inhibitor V A potent inhibitor
of caspase-1-like proteases. Blocks apoptotic cell death in human
myeloid leukemia U937 cells and blocks Peptide sequence:
etoposide-induced DNA fragmentation Z-Asp-CH2-DCB Caspase-1
Inhibitor VI A potent, cell-permeable, and irreversible inhibitor
of caspase-1 (ICE), caspase-4, and caspase-5 Peptide sequence:
Z-Tyr-Val-Ala-Asp(OMe)-CH2F* Caspase-2 Inhibitor I A cell-permeable
and irreversible inhibitor of caspase-2 (ICH-1 Peptide sequence:
Z-Val-Asp(OMe)-Val-Ala- Asp(OMe)-CH2F* Caspase-2 Inhibitor II A
reversible inhibitor of caspase-2 and caspase-3 Peptide sequence:
Ac-Leu-Asp-Glu-Ser-Asp-CHO Caspase-3/7 Inhibitor I A potent,
cell-permeable, and specific, reversible inhibitor of caspase-3 (Ki
= 60 nM) and caspase-7 (Ki = 170 nM). Peptide sequence:
5-[(S)-(+)-2- (Methoxymethyl)pyrrolidino]sul- fonylisatin Caspase-3
Inhibitor I A very potent, specific, and reversible inhibitor of
caspase-3 (IC50 = 200 pM), caspase-6, caspase-7, caspase-8, and
caspase-10. Peptide sequence: Ac-Asp-Glu-Val-Asp-CHO Caspase-3
Inhibitor I, Cell- A cell-permeable inhibitor of caspase-3, as well
as caspase-6, Permeable caspase-7, caspase-8, and caspase-10. The
C-terminal DEVD- CHO sequence of this peptide is a highly specific,
potent, and Peptide sequence: reversible inhibitor of caspase-3 (Ki
< 1 nM) that has also been Ac-Ala-Ala-Val-Ala-Leu-Leu- shown to
strongly inhibit PARP cleavage in cultured human
Pro-Ala-Val-Leu-Leu-Ala-Leu- osteosarcoma cell extracts (IC50 = 200
pM). The N-terminal Leu-Ala-Pro-Asp-Glu-Val-Asp- sequence (amino
acid residues 1-16) corresponds to the CHO hydrophobic region
(h-region) of the signal peptide of Kaposi fibroblast growth factor
(K-FGF) and confers cell-permeability to the peptide. A 5 mM (1
mg/100 .mu.l) solution of Caspase-3 Inhibitor I, Cell-permeable
(Cat. No. 235427) in DMSO is also available. Caspase-3 Inhibitor II
A potent, cell-permeable, and irreversible inhibitor of caspase-3
as well as caspase-6, caspase-7, caspase-8, and caspase-10. When
Peptide sequence: using with purified native or recombinant enzyme,
pretreatment Z-Asp(OCH3)-Glu(OCH3)-Val- with an esterase is
required. A 5 mM (250 .mu.g/75 pl) solution of Z- Asp(OCH3)-FMK
DEVD-FMK (Cat. No. 264156) in DMSO is also available Caspase-3
Inhibitor III A potent, cell-permeable, and irreversible inhibitor
of caspase-3 as well as caspase-6, caspase-7, caspase-8, and
caspase-10 Peptide sequence: Ac-Asp-Glu-Val-Asp-CMK Caspase-3
Inhibitor IV A specific inhibitor of caspase-3. This tetrapeptide
inhibitor has been used with the caspase-6 inhibitor Ac-VEID-CHO to
dissect Peptide sequence. the pathway of caspase activation in
Fas-stimulated Jurkat cells Ac-Asp-Met-Gln-Asp-CHO Caspase-3
Inhibitor V A potent, cell-permeable, and irreversible inhibitor of
caspase-3, also recognizes caspase-1. When using with purified
native or Peptide sequence: recombinant enzyme, pre-treatment with
an esterase is required Z-Asp(OMe)-Gln-Met- Asp(OMe)-CH2F*
Caspase-3 Inhibitor VII A cell-permeable, non-peptidyl
pyrroloquinoline compound that acts as a potent, reversible, and
non-competitive inhibitor of Peptide sequence: caspase-3 (IC50 = 23
nM) with 10-100-fold greater selectivity.
2-(4-Methyl-8-(morpholin-4- Shown to display higher anti-apoptotic
activity than Z-VAD-FMK ylsulfonyl)-1,3-dioxo-1,3- (Cat. No.
627610) in a model of Staurosporine- (Cat. No. 569397)
dihydro-2H-pyrrolo[3,4- induced apoptosis in human Jurkat T cells.
c]quinolin-2-yl)ethyl acetate Caspase-4 Inhibitor I A reversible
caspase-4 inhibitor Peptide sequence: Ac-Leu-Glu-Val-Asp-CHO
Caspase-4 Inhibitor I, Cell- A potent, cell-permeable, and
reversible inhibitor of caspase-4. Permeable The N-terminal
sequence (amino acid residues 1-16) corresponds to the hydrophobic
region of the signal peptide of Kaposi fibroblast Peptide sequence:
growth factor and confers cell permeability to the peptide.
Ac-Ala-Ala-Val-Ala-Leu-Leu- Pro-Ala-Val-Leu-Leu-Ala-Leu-
Leu-Ala-Pro-Leu-Glu-Val-Asp- CHO Caspase-5 Inhibitor I A potent,
cell-permeable, and irreversible inhibitor of caspase-5. Strongly
inhibits caspase-1. Also inhibits caspase-4 and caspase-8 Peptide
sequence: Z-Trp-Glu(OMe)-His-Asp(OMe)- CH2F* Caspase-6 Inhibitor I
A cell-permeable, irreversible inhibitor of caspase-6. When using
with purified native or recombinant enzyme, pretreatment with an
Peptide sequence: esterase is required
Z-Val-Glu(OMe)-Ile-Asp(OMe)-
CH2F* Caspase-6 Inhibitor II, Cell- A potent, cell-permeable, and
reversible inhibitor of caspase-6.The Permeable N-terminal sequence
(amino acids 1-16) corresponds to the hydrophobic region of the
signal peptide of Kaposi fibroblast Peptide sequence: growth factor
and confers cell permeability to the peptide
Ac-Ala-Ala-Val-Ala-Leu-Leu- Pro-Ala-Val-Leu-Leu-Ala-Leu-
Leu-Ala-Pro-Val-Glud le-Asp- CHO Caspase-8 Inhibitor I, Cell- A
potent, cell-permeable, and reversible inhibitor of caspase-8 and
Permeable Granzyme B. The N-terminal sequence (amino acids 1-16)
corresponds to the hydrophobic region of the signal peptide of
Peptide sequence: Kaposi fibroblast growth factor and confers cell
permeability to the Ac-Ala-Ala-Val-Ala-Leu-Leu- peptide
Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Ala-Pro-lle-Glu-Thr-Asp- CHO
Caspase-8 Inhibitor II A potent, cell-permeable, and irreversible
inhibitor of caspase-8 and granzyme B. Effectively inhibits
influenza virus-induced Peptide sequence: apoptosis in HeLa cells.
Also inhibits granzyme B. When using with
Z-Ile-Glu(OMe)-Thr-Asp(OMe)- purified native or recombinant enzyme,
pretreatment with an CH2F* esterase is required. A 5 mM (250
.mu.g/76 .mu.l) solution of Z-IETD- FMK (Cat. No. 218840) in DMSO
is also available. Caspase-9 Inhibitor I A potent, cell-permeable,
and irreversible inhibitor of caspase-9. May also inhibit caspase-4
and caspase-5. When using with Peptide sequence: purified native or
recombinant enzyme, pretreatment with an
Z-Leu-Glu(OMe)-His-Asp(OMe)- esterase is required. A 5 mM (250
.mu.g/72 .mu.l) solution of Z-LEHD- CH2F* FMK (Cat. No. 218841) in
DMSO is also available Caspase-9 Inhibitor II, Cell- A potent,
cell-permeable, and reversible inhibitor of caspase-9. Permeable
May also inhibit caspase-4 and caspase-5. The N-terminal sequence
(amino acids 1-16) corresponds to the hydrophobic Peptide sequence:
region of the signal peptide of Kaposi fibroblast growth factor and
Ac-Ala-Ala-Val-Ala-Leu-Leu- confers cell permeability to the
peptide Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Ala-Pro-Leu-Glu-His-Asp-
CHO Caspase-9 Inhibitor III A potent, irreversible inhibitor of
caspase-9. Reported to reduce myocardial infarct size during
reperfusion (~70 nM). Peptide sequence: Ac-Leu-Glu-His-Asp-CMK
Caspase Inhibitor I A cell-permeable, irreversible, pan-caspase
inhibitor. Inhibits Fas- mediated apoptosis in Jurkat cells and
staurosporine-induced cell Peptide sequence: death in corneal
epithelial cells. When using with purified native or
Z-Val-Ala-Asp(OMe)-CH2F* recombinant enzyme, pre-treatment with an
esterase is required. Caspase Inhibitor II A potent and reversible
pan-caspase inhibitor. Peptide sequence: Ac-Val-Ala-Asp-CHO Caspase
Inhibitor II, Cell- A cell-permeable, reversible pan-caspase
inhibitor produced by Permeable attaching the N-terminal sequence
(amino acids 1-16) of the Kaposi fibroblast growth factor signaling
peptide, which imparts Peptide sequence: cell-permeability to VAD
peptide. Ac-Ala-Ala-Val-Ala-Leu-Leu- Pro-Ala-Val-Leu-Leu-Ala-Leu-
Leu-Ala-Pro-Val-Ala-Asp-CHO Caspase Inhibitor III A cell-permeable,
irreversible, broad-spectrum caspase inhibitor. Peptide sequence:
Boc-Asp(OMe)-CH2F* Caspase Inhibitor IV A general, irreversible
caspase inhibitor. Peptide sequence: Boc-Asp(OBzl)-CMK Caspase
Inhibitor VI An irreversible general caspase inhibitor. Useful for
studies involving recombinant, isolated, and purified caspase
enzymes. Peptide sequence: Unlike Caspase Inhibitor I (Cat. No.
627610), this inhibitor does not Z-Val-Ala-Asp-CH2F* require
pretreatment with esterase for in vitro studies. A 10 mM (1 mg/221
.mu.l) solution of Caspase Inhibitor VI (Cat. No. 219011) in DMSO
is also available Caspase Inhibitor VIII A potent, reversible
inhibitor of caspase-2 (Ki = 3.5 nM), caspase-3 (Ki = 1 nM) and
caspase-7 (Ki = 7.5 nM). Also serves as an Peptide sequence:
inhibitor of DRONC (Drosophila caspase), a glutamate/aspartate
Ac-Val-Asp-Val-Ala-Asp-CHO protease. Caspase Inhibitor X A
benzodioxane containing 2,4-disubstituted thiazolo compound that
acts as a selective, reversible and competitive inhibitor of
Peptide sequence: caspases (Ki = 4.3 pM, 6.2 pM and 2.7 pM for
caspase-3, -7 and - BI-9B12 8, respectively). The benzodioxane
moiety is shown to fit in the `aspartate hole` of the caspases and
possibly disrupt caspase-8 assisted cleavage of BID, a proapoptotic
protein. Weakly affects the activity of anthrax lethal factor, a
metalloprotease, at ~20 .mu.M Caspase-1 Inhibitors Including, but
not limited to Ac-N-Me-Tyr-Val-Ala-Asp-aldehyde (pseudo acid)
Ac-Trp-Glu-His-Asp-aldehyde (pseudo acid)
Ac-Tyr-Val-Ala-Asp-aldehyde (pseudo acid)
Ac-Tyr-Val-Ala-Asp-chloromethylketone
Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone
Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetal
Ac-Tyr-Val-Lys-Asp-aldehyde (pseudo acid)
Ac-Tyr-Val-Lys(biotinyI)-Asp-2,6-dimethylbenzoyloxymethylketone
Biotinyl-Tyr-Val-Ala-Asp-chloromethylketone
Biotinyl-Val-Ala-DL-Asp-fluoromethylketone
Fluorescein-6-carbonyl-Tyr-Val-Ala-DL-Asp(OMe)- fluoromethylketone
Fluorescein-6-carbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone
Z-Asp-2,6-dichlorobenzoyloxymethylketone Z-Tyr-Val-Ala-Asp-ch
loromethylketone Z-Val-Ala-DL-Asp-fluoromethylketone
Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone Caspase-2 Inhibitors
Including, but not limited to Ac-Val-Asp-Val-Ala-Asp-aldehyde
(pseudo acid)
Fluorescein-6-carbonyl-Val-Asp(OMe)-Val-Ala-DL-Asp(OMe)-
fluoromethylketone
Z-Val-Asp(OMe)-Val-Ala-DL-Asp(OMe)-fluoromethylketone Caspase-3
Precursor Protease Including, but not limited to Inhibitors
Ac-Glu-Ser-Met-Asp-aldehyde (pseudo acid)
Ac-Ile-Glu-Thr-Asp-aldehyde (pseudo acid) Caspase-3 Inhibitors
Including, but not limited to Ac-Asp-Glu-Val-Asp-aldehyde (pseudo
acid) Ac-Asp-Met-Gln-Asp-aldehyde (pseudo acid)
Biotinyl-Asp-Glu-Val-Asp-aldehyde (pseudo acid) Caspase-3/7
Inhibitor II
Fluorescein-6-carbonyl-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-
fluoromethylketone
Z-Asp(OMe)-Gln-Met-DL-Asp(OMe)-fluoromethylketone
Z-Asp-Glu-Val-Asp-chloromethylketone
Z-Asp(OMe)-Glu(OMe)-Val-DL-Asp(OMe)-fluoromethylketone Caspase-4
Inhibitors Including, but not limited to
Ac-Leu-Glu-Val-Asp-aldehyde (pseudo acid)
Z-Tyr-Val-Ala-DL-Asp-fluoromethylketone Caspase-6 Inhibitors
Including, but not limited to Ac-Val-Glu-Ile-Asp-aldehyde (pseudo
acid) Fluorescein-6-carbonyl-Val-Glu(OMe)-Ile-DL-Asp(OMe)-
fluoromethylketone
Z-Val-Glu(OMe)-Ile-DL-Asp(OMe)-fluoromethylketone Caspase-8
Inhibitors Including, but not limited to
Ac-Ile-Glu-Pro-Asp-aldehyde (pseudo acid)
Boc-Ala-Glu-Val-Asp-aldehyde (pseudo acid)
Fluorescein-6-carbonyl-11e-Glu(OMe)-Thr-DL-Asp(OMe)-
fluoromethylketone
Fluorescein-6-carbonyl-Leu-Glu(OMe)-Thr-DL-Asp(OMe)-
fluoromethylketone
Z-Ile-Glu(OMe)-Thr-DL-Asp(OMe)-fluoromethylketone
Z-Leu-Glu(OMe)-Thr-DL-Asp(OMe)-fluoromethylketone
Z-LE(OMe)TD(OMe)-FMK Caspase-9 Inhibitors Including, but not
limited to Ac-Leu-Glu-His-Asp-aldehyde (pseudo acid)
Ac-Leu-Glu-His-Asp-chloromethylketone
Fluorescein-6-carbonyl-Leu-Glu(OMe)-His-DL-Asp(OMe)-
fluoromethylketone Caspase-10 Inhibitors Including, but not limited
to Fluorescein-6-carbonyl-Ala-Glu(OMe)-Val-DL-Asp(OMe)-
fluoromethylketone Z-Ala-Glu-Val-DL-Asp-fluoromethylketone 3.2.
Calpain Calpain Inhibitor III A potent, cell-permeable inhibitor of
calpain I and II (Ki =8 nM). Reduces capsaicin-mediated cell death
in cultured dorsal root Peptide sequence: ganglion. Reported to
block A23187-induced suppression of Z-Val-Phe-CHO neurite outgrowth
in isolated hippocampal pyramidal neurons. Exhibits neuroprotective
effect in glutamate-induced toxicity. Calpain Inhibitor IV A
potent, cell-permeable, and irreversible inhibitor of calpain II
(k2 = 28,900 M-1s-1). Also acts as an inhibitor of cathepsin L (k2
= Peptide sequence: 680,000 M-1s-1). Z-Leu-Leu-Tyr-CH2F Calpain
Inhibitor V A potent, cell-permeable, and irreversible inhibitor of
calpain Peptide sequence: Mu-Val-HPh-CH2F (Mu = morpholinoureidyl;
HPh = homophenylalanyl) Cell-permeable, peptide aldehyde inhibitor
of calpain I (Ki = 190 nM), Ac-Leu-Leu-Nle-al calpain II (Ki = 150
nM), cathepsin L (Ki = 0.5 nM) and other neutral cysteine
proteases. Inhibits cell cycle progression at G1/S and
metaphase/anaphase in CHO cells by inhibiting cyclin B degradation.
Also stimulates HMG-CoA synthase transcription by inhibiting
degradation of active SREBP-1 (sterol regulatory element-binding
protein 1). Protects against neuronal damage caused by hypoxia and
ischemia. Inhibits apoptosis in thymocytes and metamyelocytes. Also
prevents nitric oxide production by activated macrophages by
interfering with the transcription of inducible nitric oxide
synthase (iNOS; NOS II). Inhibits proteolytic degradation of
IkBalpha and IkB.beta. in RAW macrophages induced with LPS. It also
prolong association of MHC class I molecules with the transporters
associated with antigen processing Z-LLY-FMK Calpain
N-Acetyl-Leu-Leu-Met Calpain I N-Acetyl-Leu-Leu-Nle-CHO Calpain I
3.3. others BAPTA/AM Membrane-permeable form of BAPTA. Can be
loaded into a wide variety of cells, where it is hydrolyzed by
cytosolic esterases and is trapped intracellularly as the active
chelator BAPTA. Prevents cocaine-induced ventricular fibrillations.
Abolishes vitamin D3- induced increase in intracellular Ca2+.
Induces inactivation of protein kinase C. Also inhibits
thapsigargin-induced apoptosis in rat thymocytes. Granzyme B
Inhibitor I A weak inhibitor of the human and murine granzyme B.
Also inhibits the apoptosis-related DNA fragmentation in
lymphocytes Peptide sequence: by fragmentin 2, a rat lymphocyte
granule protease homologous to Z-Ala-Ala-Asp-CH2CI granzyme B (ID50
= 300 nM). Granzyme B Inhibitor II A potent, reversible inhibitor
of granzyme B and caspase-8 (Ki = 1 nM). Also inhibits caspase-1
(<6 nM), caspase-6 (5.6 nM), and Peptide sequence: caspase-10
(27 nM). Ac-Ile-Glu-Thr-Asp-CHO Granzyme B Inhibitor IV A
reversible inhibitor of granzyme B and caspase-8 Peptide sequence:
Ac-Ile-Glu-Pro-Asp-CHO Leupeptin, Hemisulfate, A reversible
inhibitor of trypsin-like proteases and cysteine Microbial
proteases. Also known to inhibit activation-induced programmed cell
death and to restore defective immune responses of HIV+ donors
N-Ethylmaleimide Sulfhydryl alkylating reagent that inhibits
H+-ATPase and suppresses the short circuit current (IC50 = 22
.mu.M) in pancreatic duct cells. Inactivates NADP-dependent
isocitrate dehydrogenase. Also a potent inhibitor of both Mg2+ and
Ca2+/Mg2+-stimulated DNA fragmentation in rat liver nuclei.
Stimulates arachidonic acid release through activation of PLA2 in
endothelial cells N.alpha.-Tosyl-Lys Chloromethyl Inhibits
trypsin-like serine proteinases. Irreversibly inactivates Ketone,
Hydrochloride (TLCK) trypsin without affecting chymotrypsin.
Prevents nitric oxide production by activated macrophages by
interfering with transcription of the iNOS gene. Blocks cell-cell
adhesion and binding of HIV-1 virus to the target cells. In
macrophages, blocks nitric oxide synthase induced by
interferon-.gamma. and lipopolysaccharides (EC50 = 80 .mu.M).
Prevents endonucleolysis accompanying apoptotic death of HL-60
leukemia cells and normal thymocytes
Omi/HtrA2 Protease Inhibitor, A cell-permeable
furfurylidine-thiobarbituric acid compound that Ucf-101 acts as a
potent, specific, competitive, and reversible inhibitor of the
pro-apoptotic, heat-inducible, mitochondrial serine protease
Omi/HtrA2 (IC50 = 9.5 .mu.M for His-Omi134-458). Shows very little
activity against various other serine proteases tested (IC50
.ltoreq. 200 .mu.M). Reported to block Omi/HtrA2 induced cell death
in caspase-9 (-/-) null fibroblasts. Phenylarsine Oxide A
membrane-permeable protein tyrosine phosphatase inhibitor (IC50 =
18 .mu.M). Stimulates 2-deoxyglucose transport in insulin-
resistant human skeletal muscle and activates p56Ick protein
tyrosine kinase. Blocks TNF-a-dependent activation of NF-KB in
human myeloid ML-la cells. PAO inhibits the protease activities of
recombinant human caspases as well as endogenous caspases that are
active in extracts of pre-apoptotic chicken DU249 cells (S/M
extracts). Phorbol-12,13-dibutyrate Strong irritant for mouse skin,
but only moderately active as a tumor promoter. Activates protein
kinase C. Stimulates the phosphorylation of Na+,K+-ATPase, thereby
inhibiting its activity. Promotes the expression of inducible NOS
in cultured hepatocytes. Commonly used in binding studies or in
applications requiring high concentrations of phorbol compounds.
Hypericin Inhibits PKC, CKII, MAP Kinase, Insulin R, EGFR, PI-3
Kinase and also noted to possess antiviral activity. Butyrolactone
I A cell-permeable and highly selective inhibitor of
cyclin-dependent protein kinases (Cdks) that inhibits cell cycle
progression at the G1/S and G2/M transitions. Inhibits
p34cdk1/cyclinB (Cdk1; IC50 = 680 nM). Also selectively inhibits
Cdk2 and Cdk5 kinases. Has little effect on casein kinase I, casein
kinase II, EGF receptor kinase, MAP kinase, PKA, and PKC. Shown to
prevent the phosphorylation of retinoblastoma protein and H1
histone. Also blocks Fas-induced apoptosis in HL-60 cells and shows
antitumor effects on human lung cancer cell lines Nilotinib
Spezifischer BCR-ABL-Tyrosinkinase-lnhibitor Quercetin(Sophoretin)
Quercetin is a PI3K and PKC inhibitor with IC50 of 3.8 .mu.M and 15
.mu.g/ml. It strongly abrogated PI3K and Src kinases, mildly
inhibited Akt1/2, and slightly affected PKC, p38 and ERK1/2. [1][2]
Quercetin is a naturally-occurring polar auxin transport inhibitor
with IC50 of 0.8, 16.7, 6.1, 11.36 .mu.M for the inhibition of LDH%
release, the inhibition of TNF-induced PMN-EC adhesion, TNF-
induced inhibition of DNA synthesis and proliferation. It is a type
of plant-based chemical, or phytochemical, known as a flavonol and
a plant-derived flavonoid found in fruits, vegetables, leaves and
grains. It also may be used as an ingredient in supplements,
beverages or foods. In several studies, it may have anti-
inflammatory and antioxidant properties, and it is being
investigated for a wide range of potential health benefits
EXAMPLES
[0182] In the following examples, materials and methods of the
present invention are provided. It should be understood that these
examples are for illustrative purpose only and are not to be
construed as limiting this invention in any manner.
I. Materials and Methods
[0183] A test system was designed, wherein cell-containing
biological samples, here whole blood samples, were incubated at
room temperature (RT) for up to 6 or 7 days. Therein, the sample
stabilizing properties of the additives of the present invention
were tested on day 0, day 3 and day 6/7 the samples. The samples
were processed according to the following protocols, where
applicable (for details, see also the specific examples in the
results section):
1. Measurement of Blood Cell Integrity by Fluorescence Activated
Cell Sorting (FACS)
1.1. Lysis of Red Blood Cells
[0184] Transfer of 2 ml blood sample into a fresh 15 ml Falcon tube
[0185] Addition of 5-fold Buffer EL (QIAGEN) [0186] Inverting of
the sample (10.times.) [0187] Incubation on ice (10 min.) [0188]
Centrifugation for 10 min. @ 400.times.g and 4.degree. C. [0189]
Discard of the supernatant [0190] Addition of 2-fold Buffer EL
(QIAGEN) to the white blood cell pellet [0191] Resolution of the
pellet in Buffer EL (QIAGEN) by slight vortexing [0192]
Centrifugation for 10 min @ 400.times.g [0193] Discard of the
supernatant [0194] Addition of 500 .mu.l FACS Flow (Becton,
Dickinson Plymouth, UK) to the white blood cell pellet [0195]
Resolution of the pellet in FACS Flow by slight vortexing [0196]
Transfer of 1 ml FACS Flow into a fresh FACS tube [0197] Transfer
of 100 .mu.l of the resolved pellet into a FACS tube
[0198] Red blood cells are lysed because otherwise, the decisive
cell populations (which can release e.g. genomic DNA) are not
distinguishable in the FACS analysis due to the high amount of red
blood cells.
1.2. Measurement of Cell Integrity by Flow Cytometry
[0199] The measurement was performed according to manufacturer's
instruction (FACSCalibur; Becton, Dickinson Plymouth, UK).
2. Separation of Blood Plasma
[0200] To separate the blood plasma from the whole blood, the blood
samples were centrifuged for 15 min at 5000 rpm, and the obtained
plasma samples were again centrifuged for 10 min at 16.000.times.g
at 4.degree. C.
[0201] The resulting blood plasma was used for isolating the
nucleic acids contained therein.
3. Nucleic Acid Purification
[0202] The circulating, extracellular nucleic acids were purified
from the obtained plasma samples using the QIAamp.RTM. Circulating
NA Kit (according to the handbook). In brief: [0203] 10 ml sample
input; [0204] lysis: 1 ml Proteinase K and 8 ml Buffer ACL (QIAGEN)
[0205] binding: 18 ml Buffer ACB (QIAGEN) [0206] wash-steps:
unchanged and according to handbook [0207] elution in 60 .mu.l
Buffer AVE (QIAGEN)
4. Analysis of the Eluates
[0208] The eluates obtained according to 3. were stored at
-20.degree. C. till all samples (including day 6/7 samples) were
purified. Afterwards, eluates of the same condition were pooled and
treated as follows:
4.1. Measurement of the blood cell stability/DNA release by the
determination of DNA size distribution using a chip gel
electrophoresis (2100 Bioanalyzer; Agilent Technologies; Inc., USA)
according to manufacturer's instruction (see handbook Agilent DNA
7500 and DNA 12000 Kit Guide), but 1.5 .mu.l instead of 1 .mu.l
sample were transferred to the wells. 4.2. DNA quantification with
a real time PCR assay, sensitive for DNA degradation (target: 500
and 66 bp long ribosomal 18S DNA coding sequences).
[0209] The DNA duplex assay was carried out according to the
QuantiTect.RTM. Multiplex PCR handbook (Qiagen) with the following
adaptions: [0210] Primer concentration was up scaled from 8 .mu.M
to 16 .mu.M. [0211] Annealing/extension step was extended from 1 to
2 min. (samples were diluted 1:10 before amplification) 4.3. RNA
detection using real time PCR assays, sensitive for variations in
circulating cell-free RNA levels (target: 18S rRNA, IL8, c-fos and
p53). The RNA assays were carried out according to the conditions
described in Tables 2 to 4.
TABLE-US-00003 [0211] TABLE 2 shows compositions of PCR reagents
and cycling conditions of the p53 mRNA one step real time PCR.
TaqMan MasterMix (MM) single master-mix component reaction (x-fold)
c p53 FAM-BHQ + HEX-BHQ x-fach 1 x 182 1 x 20.00 mastermix/reaction
RNA 5.000 / var. 5 .mu.l RNA A. dest (PCR grade) 3.813 693.9 /
25.00 .mu.l reaction volume 2x QuantiTect Probe RT-PCR MasterMix
(Puffer) 12.500 2275.0 1 x forw. primer (20 .mu.M) 0.500 91.0 400
nM rev. primer (20 .mu.M) 0.500 91.0 400 nM probe (20 .mu.M) 0.313
56.9 250 nM RNAsin (40 U/.mu.l; Promega) 0.125 22.8 0.2 U/.mu.l
MgCl2 (25 mM) 2.000 364.0 6 mM QuantiTect RT-PCR Mix (Enzym Mix)
0.250 45.5 U/.mu.l Reaction volume [.mu.l] 25.000 3640.0
Cycling:
30 min 50.degree. C.
15 min 95.degree. C.
[0212] 40 cycles
15 seq. 95.degree. C.
TABLE-US-00004 [0213] TABLE 3 shows compositions of PCR reagents
and cycling conditions of the IL8 mRNA one step real time PCR.
single master-mix final component reaction (x-fold) konc. IL8
FAM-BHQ x-fold 1 x 106 1 x 20.00 .mu.l mastermix/reaction RNA 5.000
/ var. 5 .mu.l RNA A. dest (PCR grade) 3.751 397.6 / 25.00 .mu.l
reaction volume 2x QuantiTect Probe RT-PCR MasterMix (Puffer)
12.500 1325.0 1 x forw. primer (40 .mu.M) 0.562 59.6 900 nM rev.
primer (40 .mu.M) 0.562 59.6 900 nM probe (20 .mu.M) 0.250 26.5 200
nM RNAsin (40 U/.mu.l; Promega) 0.125 13.3 0.2 U/.mu.l MgCl2 (25
mM) 2.000 212.0 6 mM QuantiTect RT-PCR Mix (Enzym Mix) 0.250 26.5
U/.mu.l Reaction volume [.mu.l] 25.000 2120.0
Cycling:
30 min 50.degree. C.
15 min 95.degree. C.
[0214] 40 cycles
15 seq. 95.degree. C.
1 min. 60.degree. C.
TABLE-US-00005 [0215] TABLE 4 shows compositions of PCR reagents
and cycling conditions of the c-fos mRNA/18S rRNA duplex real time
PCR. for single MM Chemicals CFOS reaction x-fold FAM-JOE x-fold 1x
220 17.6 .mu.l mastermix/ reaction Template 2.4 / 2.4 .mu.l RNA A.
dest 1.00 220 20 .mu.l reaction 2x QuantiTec Mastermix 10 2200
volume forw. primer (20 .mu.M); c-fos 0.900 198 rev. primer (20
.mu.M); c-fos 0.900 198 probe (10 .mu.M); c-fos 0.500 110 forw.
primer (10 .mu.M); 18 S 0.800 176 rev. primer (10 .mu.M); 18 S
0.800 176 probe (10 .mu.M); 18 S 0.800 176 RNasin (40 U/.mu.l;
Promega) 0.100 22 MgCl2 (25 mM) 1.6 352 QuantiTect RT-PCR Mix 0.2
44 (Enzym Mix) reaction volume [.mu.l] 20.0 3872
Cycling:
30 min 50.degree. C.
15 min 95.degree. C.
[0216] 40 cycles
15 sec. 95.degree. C.
1.30 min. 60.degree. C.
TABLE-US-00006 [0217] TABLE 5 summarizes the information of the
used DNA target sequences detected in 4.2 and 4.3 amplicon sequence
length target description position size [bp] position 5'-3' [nt]
dye 18 S human p12- 66 forward GCCGCTAGAGGT 22 5' Cy5- ribosomal
region GAAATTCTTG BHQ 3' DNA of reverse CATTCTTGGCAA 21 chromo-
ATGCTTTCG some probe ACCGGCGCAAGA 21 13, 14, CGGACCAGA 15, 21, 22
18 S human p12- 500 forward GTCGCTCGCTCC 22 5' FAM- ribosomal
region TCTCCTACTT BHQ 3' DNA of reverse GGCTGCTGGCAC 19 chromo-
CAGACTT some probe CTAATACATGCC 25 13, 14, GACGGGCGCTGA 15, 21, C
22
II. Performed Experiments and Results
[0218] Subsequently, the details on the performed experiments are
explained. Details to the methods used in the examples were
described above under I.
Example 1
Stabilization by the Addition of a Caspase-Inhibitor
[0219] Two different oligopeptides, Q-VD-OPh and
Z-Val-Ala-Asp(OMe)-FMK acting as broad spectrum caspase-inhibitors,
were tested:
TABLE-US-00007 TABLE 6 Tested caspase inhibitors inhibitor
moleculare name weight solubility structure Q-VD-OPH 513,49 20 mM,
add 97 .mu.l DMSO 10 mM, add 194 .mu.l DMSO 5 mM, add 388 .mu.l
DMSO ##STR00006## Z-Val-Ala- Asp(Ome)- FMK 467,49 20 mM, add 107
.mu.l DMSO 10 mM, add 214 .mu.l DMSO 5 mM, add 428 .mu.l DMSO
##STR00007## ##STR00008##
[0220] Each tested caspase inhibitor was added to whole blood
samples (20 .mu.M end concentration in 10 ml blood; blood was
collected into Vacutainer K2E Tubes; BD). The whole blood sample
was processed as described in section I, see 2. (plasma
preparation) and 3. (nucleic acid isolation).
Results of the Chip Gel Electrophoresis
[0221] The eluted circulating cell-free DNA was separated by size
using chip gel electrophoresis (for details on the method see
above, I, 4.1). FIG. 1a shows the obtained results. The DMSO
control and the K2E blood (not treated according to the teachings
of the present invention) show the same ladder-like pattern of
bands. This pattern occurs in samples where apoptosis takes place.
During apoptosis, endonucleases degrade genomic DNA at
inter-nucleosomal linker regions and produce DNA fragments of circa
180 bp or multiples of 180 bp. Thus, apoptosis occurs in samples
which show a clear ladder-like pattern. Furthermore, the strength
(darkness) of the pattern is decisive. The darker the bands, the
more genomic DNA was released from the cells and thus contaminates
the extracellular nucleic acid population.
[0222] FIG. 1a) shows that the DMSO control and the K2E blood
samples show a strong ladder-like pattern already on day 3, which
becomes even stronger on day 7. Thus, genomic DNA was released from
the cells contained in the sample and was also degraded. This
released and degraded DNA contaminates the cell-free nucleic acids
contained in the sample. Hence, no acceptable stabilisation is
achieved with these samples.
[0223] In contrast, whole blood samples treated with
Z-Val-Ala-Asp(OMe)-FMK show a reduced ladder-like pattern in
particular on day 7 compared to the controls, indicating an
inhibition of the release of genomic DNA, respectively genomic DNA
fragmentation caused by apoptosis. This effect is confirmed by the
results shown in FIG. 1b) (see below). The effect is even more
prominent in the blood samples treated with Q-VD-OPh, which show
significantly reduced ladder-like patterns already on day 3 and day
7. Thus, the release and degradation of genomic DNA is effectively
prevented, respectively reduced by the addition of the caspase
inhibitor Q-VD-OPh.
Results of the DNA Quantification
[0224] The eluted circulating cell-free DNA was also quantified
with the real time PCR assay that is sensitive for DNA degradation
(for details on the method see above, I, 4.2). FIG. 1b) shows the
effect of the tested caspase-inhibitors on the stabilisation of the
extracellular nucleic acid population (18S DNA duplex assay) within
7 days of storage at RT, here the increase in DNA.
[0225] Detection of ribosomal 18S DNA by quantitative real-time
PCR, makes it possible to calculate the x-fold increase of DNA from
day 0 to day 3 or 7 (calculation: division of day 3 (or 7) copies
by day 0 copies). Surprisingly, the results shown in FIG. 1b)
demonstrate a reduced increase of DNA when a caspase-inhibitor,
especially Q-VD-OPh, was added to whole blood samples. The
stabilising effect of Z-Val-Ala-Asp(OMe)-FMK compared to the
standard samples was more prominent on day 7, thereby confirming
the results shown in FIG. 1a).
SUMMARY
[0226] Summarizing the results of the real time PCR and the gel
electrophoresis, it was demonstrated that the addition of Q-VD-OPh
or Z-Val-Ala-Asp(OMe)-FMK inhibits DNA fragmentation and
furthermore, reduces the release of genomic DNA into blood plasma.
Thus, adding a caspase inhibitor to whole blood is effective in
stabilising the sample and in particular the extracellular nucleic
acid population even at room temperature. Thus, using the
stabilisation method according to the present invention, allows to
ship whole blood samples even at room temperature without
jeopardizing the quality of the sample. To completely prevent
release of genomic DNA also during longer storage periods, the
concentration of Q-VD-OPh may also be increased.
Example 2
Influence of Lower Concentrations of Caspase-Inhibitor Q-VD-OPh on
Blood Stability
[0227] In this example, lower concentrations of the caspase
inhibitor Q-VD-OPh was tested in combination with glucose, wherein
the glucose was added as combination partner to support that the
blood cells stay alive (by preventing cell damage). 21.4 mM glucose
and 4 .mu.M, 1 .mu.M or no Q-VD-OPh were added to 10 ml blood drawn
into BD Vacutainer tubes and stored for up to 7 days at room
temperature. The whole blood sample was processed as described in
section I, see 2. (plasma preparation) and 3. (nucleic acid
isolation).
Results of the Chip Gel Electrophoresis
[0228] The eluted DNA was separated by size using chip gel
electrophoresis (for details on the method see above, I, 4.1). FIG.
2a shows that compared to the control samples, wherein no caspase
inhibitor was added, already 1 .mu.M caspase inhibitor
significantly reduced the genomic DNA release/fragmentation on day
7. The effect is improved if 4 .mu.M caspase inhibitor is used.
Thus, already very low concentrations of the caspase inhibitor are
effective in stabilising the blood sample, in particular when
combined with a carbohydrate.
Results of the DNA Quantification
[0229] FIG. 2b shows the effects of the tested concentrations of
the caspase-inhibitor Q-VD-OPh in combination with 21 mM glucose on
the increase of genomic DNA in the plasma (18S DNA duplex assay)
within 7 days of storage at RT. The addition of Q-VD-OPh in
combination with glucose significantly reduces the release of
genomic DNA into plasma. FIG. 2b shows only a minor increase of
genomic DNA within 7 days of storage even if only 1 .mu.M Q-VD-OPH
was added to the whole blood sample for stabilisation. The addition
of 4 .mu.M Q-VD-OPh inhibits the release of genomic DNA to plasma
to a maximum of a 4-fold increase. In contrast, drawing whole blood
in K2E Tubes without stabilisation according to the present
invention leads to approximately 40-fold increase of DNA in
plasma.
[0230] Thus, also FIG. 2 b) confirms that the caspase inhibitor has
a stabilisation effect on whole blood even at low
concentrations.
Example 3
Stabilizing Blood Cells by Osmotic Effects
[0231] Surprisingly it was also found by the inventors that blood
cells can be stabilized by adding a reagent that acts as a
hypertonic medium in whole blood. Generating a hypertonic medium by
the addition of, for example, hydroxylated organic compound(s) to
whole blood results in a slight release of water from the contained
blood cells and results in increased stability by cell shrinking.
It is assumed that said cell shrinking stabilises the cells against
mechanical forces.
[0232] Dihydroxyacetone (DHA) is an intermediate product of the
fructose metabolism and its phosphate form dihydroxyacetone
phosphate (DHAP) is part of the glycolysis. DHA was tested as
hypertonic agent. Addition of this reagent sensitively forces blood
cells to shrink without damaging them. DHA was first dissolved in
PBS (purchased from SIGMA-Aldrich Kat. No: D8537) or 3.times.MOPS
(diluted from 1 litre of 10.times.MOPS: 200 mM MOPS; 50 mM NaAc, 10
mM EDTA; pH 5; assuming that an acid medium also stabilizes ccf
RNA) obtaining 4.2M solved DHA. Then 2 ml of 4.2M DHA dissolved in
Buffer PBS or buffer 3.times.MOPS were added to 10 ml of blood to
obtain a final concentration of 0.7M DHA in whole blood. The two
different solvents of DHA were compared to PAXgene.RTM. Blood DNA
tubes (QIAGEN), a state-of-the-art blood collection tube for DNA
stabilization.
Results of the FACS Analysis
[0233] The blood cell integrity was analysed using FACS (for
details on the method see above, I, 1). FIG. 3 shows the blood cell
integrity measured by flow cytometry. The Dot-Plots visualize three
different cell populations: granulocytes (1), monocytes (2) and
lymphocytes (3). The cloud (4) in the lower left field of the plot
represents the debris, mainly generated by the lysis of
erythrocytes.
[0234] The results in FIG. 3 show that blood cells collected and
stored in PAXgene.RTM. Blood DNA tubes are not distinguishable from
each other and the debris on day 6 of storage. The addition of DHA
enables a differentiation of the subpopulations of blood cells on
day 6 of storage even though these cells become smaller as a result
of the cell shrinking. This indicates that the cells contained in
the sample were stabilised by the addition of DHA.
Results of the Chip Gel Electrophoresis
[0235] The results presented in FIG. 4a also shows a stabilisation
of the blood samples by the addition of DHA, because the release of
genomic DNA is significantly lower with the DHA treated samples
than in samples stored in PAXgene.RTM. Blood DNA tubes.
Furthermore, as is evident from FIG. 4a, DHA-stabilized samples do
not show ladder-like degradation pattern suggesting that apoptosis,
respectively a degradation of DNA is efficiently prevented.
Results of the DNA Quantification
[0236] FIG. 4b shows the effect of DHA on the increase of DNA (18S
DNA duplex assay) within 6 days of storage at RT. DHA dissolved in
3.times.MOPS provided the best results, because the level of
ribosomal 18S DNA seems to remain constant till day 3 of
storage.
[0237] The division of short amplicon copy number by long amplicon
copy number (66 bp/500 bp) indicates whether the amount of detected
short or long amplicons changes over time in a similar way. A
decrease of this ratio implies a stronger release of longer rather
than of shorter DNA molecules and can be interpreted as release of
high molecular weight genomic DNA from blood cells. The diagram
shown in FIG. 4b indicates the release of genomic DNA for all three
conditions. The results show that the presence of DHA slows this
process down. Thus, also this experiment shows that the addition of
DHA to whole EDTA blood stabilizes blood cells and hence preserves
the ccfDNA population in the cell-free plasma fraction and avoids
contaminations with DNA released from the cells contained in the
sample e.g. due to mechanical breakup.
Example 4
Testing Different Concentrations of Dihydroxyacetone
[0238] In this example, the stabilising effect of different
concentrations of DHA (0.7M, 0.5M and 0.2M) was tested.
Results of the FACS Analysis
[0239] FIG. 5 shows the blood cell integrity measured by flow
cytometry. The Dot-Plots visualize three different cell
populations: granulocytes (1), monocytes (2) and lymphocytes (3).
The cloud in the lower left field of the plot represents the
debris, mainly caused by the lysis of erythrocytes.
[0240] Due to the addition of DHA to whole blood the different cell
populations can be distinguished even on day 6 of storage
regardless of the DHA concentration. Although the results of the
flow cytometry analysis (FIG. 5) do not show differences in cell
integrity between the different concentrations of DHA
Results of the Chip Gel Electrophoresis
[0241] The results presented in FIG. 6a also show a stabilisation
of the blood samples by the addition of the different
concentrations of DHA, because the release of genomic DNA and the
degradation of the DNA is efficiently prevented.
Results of the DNA Quantification
[0242] FIG. 6b shows the effect of different DHA concentrations on
the increase of DNA (18S DNA duplex assay) within 6 days of storage
at RT. As shown in FIG. 6b, 0.5M DHA in whole blood prevents most
efficiently the release of genomic DNA. Furthermore, the ratio of
short to long amplicon copy numbers stays constant for up to 3 days
and only decreases slightly till day 6. These results demonstrate
the remarkable effect of the hypertonic agent DHA on the
stabilisation of whole blood.
Example 5
Combination of an Apoptosis Inhibitor, an Osmotically Active
Compound and an Anticoagulant
[0243] An increase of EDTA in blood collection tubes inhibits
micro- and macroclotting as it is known for PAXgene.RTM. Blood DNA
tubes. Hence, higher concentrations of EDTA may support
stabilization of blood cells and extracellular nucleic acids in
plasma. Furthermore, the experiments presented above show an
inhibitory effect of the caspase inhibitor, in particular Q-VD-OPh,
and the osmotically active compound DHA on blood cell damage and in
particular show that an increase of genomic DNA, in particular
fragmented genomic DNA, in the extracellular nucleic acid
population is efficiently reduced. Surprisingly, the caspase
inhibitors tested also prevented/inhibited the leakage of genomic
DNA into the cell-free (plasma) fraction. Hence, the combination of
these reagents results in an improved stabilization of
extracellular nucleic acids, in particular extracellular DNA, in
whole blood that lasts at least for 6 days, and furthermore,
results in an efficient stabilization of blood cells, thereby
preventing the release of genomic DNA, what otherwise would result
in a dilution of the natural extracellular nucleic acid level in
plasma.
[0244] In this example, DHA was dissolved in 2 ml 3.times.MOPS (3M
DHA in 2 ml 3.times.MOPS), 50 mg K.sub.2EDTA and 2.4 .mu.l of 5 nM
Q-VD-OPh were added and then transferred into 10 ml whole blood,
that was collected in K2E Tubes. Plasma samples were centrifuged
for 10 min at 16.000.times.g, 4.degree. C. and then purified using
the QIAamp.RTM. Circulating NA Kit (Qiagen) (details are described
above in section I).
Results of the FACS Analysis
[0245] FIG. 7a shows the blood cell integrity measured by flow
cytometry. The Dot-Plots visualize three different cell
populations: granulocytes (1), monocytes (2) and lymphocytes (3).
The cloud in the lower left field of the plot represents the
debris, mainly caused by the lysis of remaining erythrocytes.
[0246] The addition of the caspase inhibitor, the hypertonic agent
and the complexing agent to whole blood resulted in a
distinguishable pattern of blood cell populations after 6 days of
storage. Thus, the cells contained in the blood sample were
efficiently stabilised.
Results of the DNA Quantification
[0247] FIG. 7b shows the effect of the combination of EDTA, DHA and
the caspase-inhibitor Q-VD-OPH on the increase of DNA (18S DNA
duplex assay) within 6 days of storage at RT. The results indicate
that the combination of EDTA, DHA and Q-VD-OPH leads to a
remarkably strong stabilization of extracellular DNA in plasma
(level of measured 18S rDNA remains constant till day 6) and to a
strong prevention of the release of genomic DNA from blood cells
(ratio of short to long amplicon copy numbers remains constant)
till day 3 of storage. Only a slight increase of genomic DNA into
plasma becomes visible between day 3 and day 6 of storage.
[0248] Thus, the tested combination of stabilising agents is
particularly efficient in stabilising whole blood samples.
Example 6
Effect of an Apoptosis Inhibitor, an Osmotically Active Compound
and a Preventing Agent on Free Circulating RNA in Whole Blood
[0249] As a combination of K.sub.2EDTA, Q-VD-OPh and DHA showed
remarkable stabilizing effects on free circulating DNA and the
integrity of blood cells in whole blood, the stabilising capacities
of these agents on free circulating RNA was also analysed. To
preserve a constant level of free circulating RNA in plasma (as
present when collecting the blood), the stabilizing reagent(s)
should not only protect RNAs from degradation and prevent the
release of RNAs from decaying blood cells, but should also inhibit
the metabolic pathways, respectively have the effect that changes
in the metabolic pathway do not affect the extracellular RNA plasma
level, respectively should reduce respective effects. Hence
experiment 5 was repeated and the level of mRNA was measured by
real time RT-PCR.
[0250] FIG. 8 shows the effect of the combination of EDTA, DHA and
the tested caspase-inhibitor on the transcript level in plasma
within 6 days of storage. In order to measure variations in RNA
levels, target mRNAs were referred to as reference target (18S
rRNA) by calculating a .DELTA.Ct between p53, IL8 or c-fos and the
internal standard (18S rRNA). Subtracting the .DELTA.Ct of day 3 or
6 samples with the .DELTA.Ct of day 0 samples defines the
.DELTA..DELTA.Ct visualizing a relative decrease (- values) or
increase (+ values) of mRNA transcript levels. IL8 and c-fos are
genes whose transcription is induced after blood draw. Therefore,
transcript levels of these targets would rise dramatically when
cells release their contents; the addition of the stabilizing
solution according to the preferred embodiment of present invention
(combination of elevated EDTA, dihydroxyacetone, caspase inhibitor
Q-VD-OPh) strongly prevents nucleic acid release from blood cells
till day 3 of storage. But the data in the diagram above
show--surprisingly--no significant increase of c-fos and IL8 mRNA
till day 6 of storage. Thus, apparently the stabilization prevents
the degradation of RNA (p53) and the release of mRNA
(IL8/c-fos)
[0251] The transcription of p53 is repressed during continued
metabolism after blood draw and, hence, a degradation or
down-regulation of p53 mRNA would result in a decrease of
(-).DELTA. .DELTA.cts. However, the results show that the tested
QGN stabilisation solution prevents the p53 mRNA-level from being
degraded during whole blood storage for up to 6 days.
[0252] This experiment demonstrated that the addition of a
combination of elevated EDTA, dihydroxyacetone, caspase inhibitor
Q-VD-OPh to freshly drawn whole blood acts to preserve the
circulating plasma mRNA population which was present at the time of
blood draw, reducing mRNA-specific changes in mRNA concentration.
This is of particular importance for the analysis of circulating
mRNA in plasma, e.g., for identification and characterization of
potential tumor-specific mRNA species. Such studies require that
the mRNA population in plasma remains substantially unchanged
between blood draw and nucleic acid extraction and analysis.
Example 7
Stabilisation by the Addition of Dimethylacetamide (DMAA)
[0253] Two different concentrations of DMAA along with K.sub.2EDTA
were tested and compared to EDTA alone (K2E BD; 18 mg
K.sub.2EDTA).
[0254] DMAA was added to replicates of whole blood samples (0.75%
and 1.5% end concentration in 10 ml blood; blood was collected into
Vacutainer K2E Tubes; BD).
[0255] Blood samples were incubated for up to 6 days at room
temperature. On day 0, 3 and 6, whole blood samples were
centrifuged at 1912.times.g for 15 min at room temperature,
followed by a centrifugation of the plasma samples at
16.000.times.g for 10 min at 4.degree. C. 1 ml of the sample input
was used for DNA isolation following the protocol described in the
materials & methods section. DNA was eluted in 80 .mu.l EB
buffer and quantified with the RT PCR assay described in I,
4.2.
Results of the DNA Quantification
[0256] FIG. 11 shows the effects of the tested concentrations of
DMAA on the increase of genomic DNA in the plasma. Addition of DMAA
significantly reduces the release of genomic DNA into plasma. The
more DMAA is added to whole blood, the less DNA is released. Only a
minor increase of cell-free DNA within 6 days of storage was
observed if 1.5% DMAA was added to the whole blood sample.
Furthermore, as the addition of 1.5% DMAA stabilizes cell-free DNA
levels in whole blood samples more efficiently than 0.75% and the
ratio of short to long measured 18S DNA copies decreases from day 0
to day 6, higher DMAA concentrations of than 1.5% can result in
more efficient stabilization effects.
[0257] In summary, the addition of DMAA reduces the release of
genomic DNA into blood plasma. Thus, adding DMAA to a blood sample
is effective in stabilising the sample even at room
temperature.
Example 8
Influence of Sugar Alcohols on Preserving the ccfDNA Status in
Whole Blood
[0258] 10 ml whole blood samples of two donors were first collected
in BD Vacutainer K2E-EDTA (4.45 mM EDTA=Reference). Afterwards, 2
ml of the following stabilization solutions were added (given
concentrations represent final concentration in stabilized blood
solution):
TABLE-US-00008 Stabilization Inositol Maltitol Mannitol Sorbitol
DHA None Solution (M) (M) (M) (M) (M) (K2E) 1 0.1 2 0.05 3 0.01 4
0.1 5 0.05 6 0.01 7 0.1 8 0.05 9 0.01 10 0.1 11 0.05 12 0.01 13 0.5
14 X
[0259] The respectively stabilized samples were incubated at room
temperature for up to six days. On day 0, day 3 and day 6,
replicates were processed as follows. The samples were centrifuged
at 3.000 rpm for 10 minutes at room temperature in order to collect
plasma. The collected plasma was centrifuged at 16,000.times.g for
10 minutes at 4.degree. C. The cleared plasma fraction was
collected and the extracellular nucleic acids were isolated using
the QIAamp Circulating nucleic acid kit (1 ml input material, 60
.mu.l elution volume). The results are shown in relative change
compared to the test time point 0 days (day X copies/day 0 copies)
in FIG. 10. Values that are close to 1 imply preserved levels of
ccfDNA. The higher the value, the less stabilization is achieved.
From the sugar alcohols tested, very good results were achieved
with DHA (0.5 M). Here, the lowest increase of ccfDNA levels in the
plasma fraction was observed. Other suitable alternatives are
inositol in concentrations of for example 0.05 M and maltitol in
concentrations .ltoreq.0.01 M. Furthermore, stabilization effects
over 3 days were also seen with mannitol.
Example 9
Influence of DMAA, DHA and Glycine on ccfDNA Level
[0260] 10 ml whole blood samples of three donors were first
collected in BD Vacutainer K2E-EDTA (4.45 mM EDTA=reference).
Afterwards, 2 ml of the following solutions were added (given
concentrations represent final concentration in stabilized blood
solution):
TABLE-US-00009 OPH Stabilization DHA DMAA (caspase None Solution
(M) (%) EDTA inhibitor) (K2E) 1 0.5 2 0.1 3 0.05 4 (QGN mixture)
0.5 14 mM 1 .mu.M 5 1% 14 mM 1 .mu.M 6 3% 14 mM 1 .mu.M 7 5% 14 mM
1 .mu.M 8 X
[0261] The samples were processed as described in example 8. The
results as well as the test conditions are shown in FIG. 11. As can
be seen, DHA alone stabilizes the level of ccfDNA for up to three
days (see donor 1). Particularly stable ccfDNA levels were obtained
when using the QGN mixture. Results comparable to the QGN mixture
could be obtained when adding DMAA to the sample in combination
with e.g. increasing the EDTA concentrations and adding a caspase
inhibitor.
Example 10
Influence of Sugar Alcohol in Combination with Caspase Inhibitor
and Increased EDTA Concentrations on ccfDNA Level
[0262] 10 ml whole blood samples of two donors were first collected
in the BD Vacutainer K2E-EDTA (4.45 mM EDTA=reference). Then, 2 ml
of the following solutions were added (given concentrations
represent final concentration in stabilized blood solution):
1: 0.5 M DHA, 1 .mu.M OPH, 14 mM EDTA (QGN mixture); 2: 0.5 M
Inositol in QGN mix (without DHA); 3: 0.01 M Maltitol in QGN mix
(without DHA).
[0263] The samples were then processed as described in example 8.
The results are shown in FIG. 12. The best results were obtained
for the QGN mixture. Combinations of sugar alcohol to the QGN
mixture also showed stabilizing effects when compared to the EDTA
stabilized samples.
Example 11
Influence of Combinations of DMAA and OPH (Caspase Inhibitor)
Concentrations on ccfDNA Levels
[0264] 10 ml whole blood samples were first collected in BD
Vacutainer K2E-EDTA (4.45 mM EDTA=reference). Then, 2 ml of the
following solutions were added (given concentrations represent
final concentration in stabilized blood solution). Each condition
was tested with six tubes from different donors.
1: EDTA reference (BD Vacutainer K2E); 2: QGN mixture;
3: 50 mg EDTA, 1 .mu.M OPH;
4: 50 mg EDTA, 2 .mu.M OPH;
5: 50 mg EDTA, 1 .mu.M OPH, 5% DMAA;
6: 50 mg EDTA, 1 .mu.M OPH, 10% DMAA;
7: 50 mg EDTA, 2 .mu.M OPH, 5% DMAA;
8: 50 mg EDTA; 2 .mu.M OPH and 10% DMAA.
[0265] The sample incubation, isolation of plasma and isolation
from nucleic acids from the cleared plasma fraction were performed
as described in example 8. However, after the first centrifugation
step at 3.000 rpm, plasma samples of identical stabilization
conditions were pooled before the second centrifugation step for
plasma clearing (16.000.times.g) was carried out. The results are
shown in FIG. 13. As can be seen, different DMAA concentrations in
combination to different OPH concentrations show comparable results
to the QGN mixture. FIG. 14 shows the influence of combinations of
DMAA and OPH concentrations on ccfDNA levels (different scaling due
to exclosure of reference data).
Example 12
Influence of Combination of QGN Mixture with Sugar Alcohols on
ccfDNA Level
[0266] 10 ml whole blood samples were collected in BD Vacutainer
K2E-EDTA (4.45 mM EDTA=reference). Afterwards, 2 ml of the
following solutions was added (given concentrations represent final
concentration in stabilized blood solution). Each condition was
tested with six tubes and thus six different donors.
1: EDTA reference (BD Vacutainer K2E); 2: QGN mixture (0.01 M DHA,
14 mM EDTA, 1 .mu.M OPH);
3: 0.01M DHA;
4: 5% DMAA, 14 mM EDTA, 1 .mu.M OPH;
5: 0.01M DHA, 3% DMAA, 1 .mu.M OPH, 14 mM EDTA;
6: 1 .mu.M OPH, 14 mM EDTA, 0.01 M DHA, 0.01 M Maltitol;
7: 1 .mu.M OPH, 14 mM EDTA, 0.01 M DHA, 0.05 M Inositol;
8: 1 .mu.M OPH, 14 mM EDTA, 0.01 M DHA, 0.05M Inositol, 0.01 M
Maltitol.
[0267] The samples were processed as described in example 11.
However, the samples were not stored at room temperature, but at
37.degree. C. instead. The results are shown in FIG. 15. As can be
seen, stable levels of ccfDNA were achieved, especially when 5%
DMAA was added in combination with 14 mM EDTA and 1 .mu.M OPH.
Therefore, unexpectedly, a very good stabilization of ccfDNA in
whole blood could be achieved even if at elevated temperatures
(37.degree. C.).
Example 13
Incubation at 37.degree. C.--Analysis of Single Donor Samples
[0268] Whole blood samples from six different donors were collected
in BD Vacutainers K2E, and then 2 ml of the following stabilization
solutions were added per 10 ml whole blood (given concentrations
represent final concentration in stabilized blood solution):
1: 2 .mu.M OPH, 14 mM EDTA, 5% DMAA;
2: 1 .mu.M OPH, 14 mM EDTA, 3% DMAA;
3: 1 .mu.M OPH, 14 mM EDTA, 0.01 M DHA, 3% DMAA.
[0269] The samples were incubated at 37.degree. C. for up to six
days. Otherwise, the same procedure as in example 8 was followed.
The results are shown in FIGS. 16 and 17. As can be seen, for all
six donors, the level of ccfDNA was preserved when different
concentrations of DMAA in combination with OPH and EDTA were added
to the blood samples. Therefore, an efficient stabilization can be
achieved with the method according to the present invention.
Example 14
Limit of Detection (LoD)
[0270] Extracellular nucleic acids are often comprised in very
small amounts in the sample. Therefore, it is important to have a
stabilization procedure which not only efficiently preserves the
extracellular nucleic acids within the stabilized sample, but
additionally allows to subsequently isolate the extracellular
nucleic acids with high yield from the stabilized sample. Example
14 demonstrates that the stabilization method according to the
present invention is superior to prior art stabilization methods in
that the extracellular nucleic acids can be isolated with higher
yield from the stabilized samples. This advantageously reduces the
limit of detection and thus, allows to reliably determine also rare
target nucleic acids within the population of extracellular nucleic
acids.
[0271] The following stabilization solutions/tubes were compared:
[0272] 1. Cell-free RNA BCT (Streck Inc, cat. #:218976--comprises a
formaldehyde releaser as stabilizer) [0273] 2. BD Vacutainer
K.sub.2E (BD, Cat. #: 367525--comprises EDTA)=reference [0274] 3.
QGN stabilization (5% DMAA, 14 mM EDTA, 2 .mu.M OPH (caspase
inhibitor))
[0275] Whole blood samples were collected in cell-free RNA BCT and
BD Vacutainer K2E tubes. To one half of blood collected in BD
tubes, the QGN stabilization solution was added. Thus, the sample
stabilized according to the invention comprise an additional amount
of EDTA that is contributed by the BD Vacutainer stabilization. The
samples were centrifuged at 3.000.times. rpm for 10 minutes, and
the obtained plasma was aliquoted to 1.5 ml replicates. Afterwards,
the following amounts of DNA spike-in control (1.000 bp) were added
per sample: 1.000 copies, 5000 copies, 100 copies, 50 copies and 10
copies.
[0276] 8 replicates of 500 to 10 copies/sample, 4 replicates of
1.000 copies/sample and 5 copies/sample were prepared. The samples
were incubated for 3 days at room temperature. The sample
preparation was done on the QIAsymphony SP automated system, using
the QIAsymphony virus/bacteria Cell-free 1000 application which
allows isolating extracellular nucleic acids from plasma samples.
The nucleic acids were eluted in 60 .mu.l; the subsequent PCR was
performed in triplicates.
[0277] The results are shown in FIG. 18. As can be seen, 100%
hit.gtoreq.1.000 copies per sample was obtained when using either
the BD EDTA tubes or the stabilization solution according to the
present invention. This shows that the isolation of nucleic acids
is not impaired when using the stabilization solution according to
the present invention. In contrast, the stabilization that is based
on the use of a formaldehyde releaser (Streck) shows a strong
inhibition of the nucleic acid isolation. As can be seen,
significantly less nucleic acids could be isolated from the
respective samples, even with those samples wherein 500 or even
1.000 copies were spiked in. Furthermore, FIG. 18 shows that the
best sensitivity was obtained with a sample stabilized according to
the present invention. Even for those embodiments wherein only 10
copies per sample were spiked in, still 13% positive PCR hits were
obtained. Thus, the method according to the present invention not
only efficiently stabilizes the samples such as blood samples but
furthermore allows the subsequent recovery of even very
low-abundant extracellular nucleic acids. This is an important
advantage because it makes this method particularly suitable for
diagnostic applications and e.g. the detection of rare target
extracellular nucleic acids such as e.g. tumor derived
extracellular nucleic acids or fetal nucleic acids. In particular,
in the lower copy numbers, the stabilization solution that is based
on the use of formaldehyde releasers had a very low performance and
showed the highest limit of detection.
[0278] This is also confirmed by the following table:
TABLE-US-00010 95% confidence Dose for interval DNA centile 95 min
max Fragment Tube/stabilizing [copies] [copies] [copies] 1000 bp BD
K2E 386 230 995 Streck RNA 9902 2909 164606 QGN 599 319 1749
[0279] As can be seen from said table, for the 1.000 bp fragment,
the results achieved with EDTA sample and the stabilization
solution of the present invention is comparable. Thus, the
stabilization according to the invention does not impair the
subsequent isolation of nucleic acids. Stabilization using a
formaldehyde releaser showed the highest limit of detection and
thus demonstrates that the subsequent isolation of the nucleic acid
was strongly impaired. Therefore, the stabilization according to
the present invention is suitable for sensitive detection of rare
ccfDNA targets, which is not achieved by using state of the art
methods.
[0280] This is also confirmed by the results shown in FIGS. 19 and
20. As can be seen, comparable ccfDNA yields are obtained for EDTA
stabilized samples and samples stabilized using the method
according to the present invention (measured by 18 S rDNA qPCR).
However, reduced ccfDNA yields were obtained for the stabilization,
which involves the use of formaldehyde releasers (Streck tubes).
The yield of formaldehyde stabilized samples was reduced by
approximately 50% compared to the EDTA stabilized samples. In
contrast, the stabilization reagent according to the present
invention has no adverse effect on ccfDNA yield, when using
conventional nucleic acid isolation methods. This is an important
advantage as it allows to integrate the stabilization method
according to the present invention into existing nucleic acid
isolation procedures and workflows.
Example 15
Spike-in of 10 4 IU/ml HIV, HCV to Whole Blood Samples of 3
Donors
[0281] Whole blood samples were collected in BD Vacutainer 2KE
tubes. Afterwards 2 ml of the following stabilization solution was
added (given concentrations represent final concentration in
stabilized blood solution). Then, HIV and HCV were added to the
whole blood samples at 10 4 IU/ml.
1: 5% DMAA, 50 mg EDTA, 1 .mu.M OPH, 0.05 M Inositol;
2: 5% DMAA, 50 mg EDTA, 1 .mu.M OPH, 0.01 M Maltitol;
3: 5% DMAA, 50 mg EDTA, 1 .mu.M OPH, 0.05 M Inositol; 0.01 M
Maltitol;
4: 2% Inositol, 4% Sorbitol.
[0282] BD Vacutainer K2E stabilized samples served again as
reference.
[0283] The samples were incubated at room temperature for up to six
days at 37.degree. C. On day 0 and day 3, replicates were processed
as follows: the samples were centrifuged at 3.000 rpm, for 15
minutes at room temperature to collect the plasma. The obtained
plasma was then again centrifuged at 16.000.times.g for 10 minutes,
at 4.degree. C. Extracellular nucleic acids obtained from the
cleared plasma supernatant was purified using the QIAsymphony
virus/bacteria Cell-free 1000 protocol. 1 ml plasma was used as
input material, 60 .mu.l volume was used for elution. The results
are shown in FIG. 21. As can be seen, combining DMAA, EDTA and OPH
with sugar alcohols allows to stabilize viral nucleic acids levels
for up to three days at 37.degree. C. Therefore, the method
according to the present invention is particularly suitable for
diagnostic applications and is also suitable for stabilizing the
samples in environments wherein potentially no refrigerating
facilities are available. .DELTA.Ct between day 0 and day 3 is
reduced (.DELTA.Ct of approximately 2.5 to .DELTA.Ct of
approximately 1) compared to the EDTA blood reference. Furthermore,
stabilization effects were seen with a combination of Sorbitol in
combination with Inositol (.DELTA.Ct of approximately 1 to
1.4).
[0284] FIG. 22 shows the decrease of HCV in whole blood that was
incubated at 37.degree. C. Again, it is shown that when combining
DMAA, EDTA and OPH with sugar alcohols, the HCV nucleic acid level
is stabilized, indicated by a slowed decline in viral RNA levels,
for three days at 37.degree. C. .DELTA.Ct between day 0 and day 3
is reduced (.DELTA.Ct of approximately 1) compared to the EDTA
blood reference (.DELTA.Ct of approximately 2-3). Furthermore, good
stabilizing effects were achieved for Sorbitol in combination with
Inositol.
Example 16
Stabilization with N,N Dimethylpropanamid and Caspase Inhibitor
[0285] Blood from two different donors was collected into 10 ml K2
EDTA tubes (BD). 4.5 ml of the respectively collected blood was
mixed with 0.9 ml stabilization solution containing (per ml of
stabilization solution): 34.2 mg K2 EDTA, 1.2 ml
Quinoline-Val-Asp-CH2-OPH (caspase inhibitor) solution (1 mg
dissolved in 388 .mu.l DMSO) and 0.15 g or 0.3 g, or 0.45 g N,N
dimethylpropanamide or 0.3 ml DMAA, respectively. Thereby, the
following final concentration in the blood/stabilization mixture
was obtained which is as follows:
5.7 mg K2 EDTA, 1 .mu.M Quinoline-Val-Asp-CH2-OPH (caspase
inhibitor) and 2.5, 5 or 7.5% (w/v) NN dimethylpropanamide or 5%
(v/v) DMAA, respectively.
[0286] All stabilized blood samples were set up in triplicates per
condition and test time point. At time point 0 (reference),
immediately after mixing the stabilization solution and blood,
plasma was generated and the ccfDNA was extracted. The residual
blood sample was stored for three days and six days at room
temperature. As a control, the EDTA stabilized blood sample was
also stored for 3 and 6 days. The plasma was generated from the
stabilized and unstabilized (EDTA) blood samples by inverting the
blood containing tubes for four times. Then, the tubes were
centrifuged for 15 minutes at 3.000 rpm/1912.times.g. 2.5 ml of the
plasma fraction was transferred into a fresh 15 ml falcon tube and
centrifuged for 10 minutes at 16.000.times.g. 2 ml of the
respectively cleared plasma was used for isolating the
extracellular nucleic acid using the QIAamp circulating nucleic
acid kit.
[0287] The results are shown in shown in FIGS. 23 and 24. Shown is
the increase of DNA relative to time point 0 with 2.5%, 5% and 7.5%
N,N dimethylpropanamide or 5% DMAA (fold change) using different
amplicon lengths of 18SrRNA gene. Bars indicate the mean of the
triplicate samples per condition and test time point. All solutions
according to the present inventions show significantly lower
amounts of released DNA after storage for 3 and 6 days at room
temperature compared to the unstabilized EDTA blood.
Sequence CWU 1
1
7215PRTUnknownBax-Inhibiting peptide, V5 1Val Pro Met Leu Lys 1 5
26PRTUnknownSTAT3 Inhibitor peptide 2Pro Tyr Leu Lys Thr Lys 1 5
34PRTArtificialGroup III Caspase Inhibitor I 3Ile Glu Pro Asp 1
44PRTArtificialCaspase 3, 7 inhibitor 4Asp Glu Val Asp 1
54PRTArtificialCaspase 8 inhibitor 5Leu Glu Thr Asp 1
64PRTArtificialCaspase 1, 4 inhibitor 6Tyr Val Ala Asp 1
74PRTArtificialCaspase 10 inhibitor 7Ala Glu Val Asp 1
84PRTArtificialCaspase 12 inhibitor 8Ala Thr Ala Asp 1
94PRTArtificialCaspase 4 inhibitor 9Leu Glu Val Asp 1
104PRTArtificialCaspase 13 inhibitor, reversible 10Leu Glu Glu Asp
1 114PRTArtificialCaspase 13 inhibitor, irreversible 11Leu Glu Glu
Asp 1 124PRTArtificialinhibitor of anti-APO-1 induced apoptosis in
L929-APO-1 cells 12Tyr Val Ala Asp 1 1320PRTArtificialCaspase 1
Inhibitor I, cell-permeable 13Ala Ala Val Ala Leu Leu Pro Ala Val
Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Tyr Val Ala Asp 20
144PRTArtificialCaspase 1 Inhibitor II, cell permeable 14Tyr Val
Ala Asp 1 154PRTArtificialCaspase 1 inhibitor IV, cell permeable
15Tyr Val Ala Asp 1 164PRTArtificialCaspase 1 Inhibitor VI, cell
permeable 16Tyr Val Ala Asp 1 175PRTArtificialCaspase 2 Inhibitor I
17Val Asp Val Ala Asp 1 5 185PRTArtificialCaspase 2 Inhibitor II
18Leu Asp Glu Ser Asp 1 5 194PRTArtificialCaspase 3 Inhibitor I
19Asp Glu Val Asp 1 2020PRTArtificialCaspase 3 Inhibitor I, cell
permeable 20Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu
Ala Pro 1 5 10 15 Asp Glu Val Asp 20 214PRTArtificialCaspase 3
Inhibitor II 21Asp Glu Val Asp 1 224PRTArtificialCaspase 3
Inhibitor III 22Asp Glu Val Asp 1 234PRTArtificialCaspase 3
Inhibitor IV 23Asp Met Gln Asp 1 244PRTArtificialCaspase 3
Inhibitor V 24Asp Gln Met Asp 1 254PRTArtificialCaspase 4 Inhibitor
I 25Leu Glu Val Asp 1 2620PRTArtificialCaspase 4 Inhibitor I, cell
permeable 26Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu
Ala Pro 1 5 10 15 Leu Glu Val Asp 20 274PRTArtificialCaspase 5
Inhibitor I 27Trp Glu His Asp 1 284PRTArtificialCaspase 6 Inhibitor
I 28Val Glu Ile Asp 1 2920PRTArtificialCaspase 6 Inhibitor II, cell
permeable 29Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu
Ala Pro 1 5 10 15 Val Glu Ile Asp 20 3020PRTArtificialCaspase 8
Inhibitor I, cell permeable 30Ala Ala Val Ala Leu Leu Pro Ala Val
Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Ile Glu Thr Asp 20
314PRTArtificialCaspase 8 Inhibitor II 31Ile Glu Thr Asp 1
324PRTArtificialCaspase 9 Inhibitor I 32Leu Glu His Asp 1
3320PRTArtificialCaspase 9 Inhibitor II, cell permeable 33Ala Ala
Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15
Leu Glu His Asp 20 344PRTArtificialCaspase 9 Inhibitor III 34Leu
Glu His Asp 1 3519PRTArtificialPan-Caspase Inhibitor II, cell
permeable 35Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu
Ala Pro 1 5 10 15 Val Ala Asp 365PRTArtificialCaspase Inhibitor
VIII 36Val Asp Val Ala Asp 1 5 374PRTArtificialcaspase 1 inhibitor
37Tyr Val Ala Asp 1 384PRTArtificialcaspase 1 inhibitor 38Trp Glu
His Asp 1 394PRTArtificialcaspase 1 inhibitor 39Tyr Val Ala Asp 1
404PRTArtificialcaspase 1 inhibitor 40Tyr Val Ala Asp 1
414PRTArtificialcaspase 1 inhibitor 41Tyr Val Ala Asp 1
424PRTArtificialcaspase 1 inhibitor 42Tyr Val Lys Asp 1
434PRTArtificialcaspase 1 inhibitor 43Tyr Val Ala Asp 1
445PRTArtificialcaspase 2 inhibitor 44Val Asp Val Ala Asp 1 5
455PRTArtificialcaspase 2 inhibitor 45Val Asp Val Ala Asp 1 5
464PRTArtificialcaspase 3 precursor inhibitor 46Glu Ser Met Asp 1
474PRTArtificialcaspase 3 precursor inhibitor 47Ile Glu Thr Asp 1
484PRTArtificialcaspase 3 inhibitor 48Asp Glu Val Asp 1
494PRTArtificialcaspase 3 inhibitor 49Asp Met Gln Asp 1
504PRTArtificialCaspase 3/7 Inhibitor II 50Asp Gln Met Asp 1
514PRTArtificialCaspase 3/7 Inhibitor II 51Asp Glu Val Asp 1
524PRTArtificialCaspase 3/7 Inhibitor II 52Asp Glu Val Asp 1
534PRTArtificialcaspase 4 inhibitor 53Leu Glu Val Asp 1
544PRTArtificialcaspase 4 inhibitor 54Tyr Val Ala Asp 1
554PRTArtificialcaspase 6 inhibitor 55Val Glu Ile Asp 1
564PRTArtificialcaspase 6 inhibitor 56Val Glu Ile Asp 1
574PRTArtificialcaspase 8 inhibitor 57Ile Glu Pro Asp 1
584PRTArtificialcaspase 8 inhibitor 58Ala Glu Val Asp 1
594PRTArtificialcaspase 8 inhibitor 59Ile Glu Thr Asp 1
604PRTArtificialcaspase 8 inhibitor 60Leu Glu Thr Asp 1
614PRTArtificialcaspase 9 inhibitor 61Leu Glu His Asp 1
624PRTArtificialcaspase 9 inhibitor 62Leu Glu His Asp 1
634PRTArtificialcaspase 10 inhibitor 63Ala Glu Val Asp 1
644PRTArtificialGranzyme B Inhibitor II 64Ile Glu Thr Asp 1
654PRTArtificialGranzyme B Inhibitor IV 65Ile Glu Pro Asp 1
6622DNAArtificialhuman ribosomal DNA forward primer 66gccgctagag
gtgaaattct tg 226721DNAArtificialhuman ribosomal DNA reverse primer
67cattcttggc aaatgctttc g 216821DNAArtificialhuman ribosomal DNA
probe 68accggcgcaa gacggaccag a 216922DNAArtificialhuman ribosomal
DNA forward primer 69gtcgctcgct cctctcctac tt
227019DNAArtificialhuman ribosomal DNA reverse primer 70ggctgctggc
accagactt 197125DNAArtificialhuman ribosomal DNA probe 71ctaatacatg
ccgacgggcg ctgac 257218DNAArtificialhuman ribosomal DNA forward
primer 72gaattgacgg aagggcac 18
* * * * *