U.S. patent application number 10/610553 was filed with the patent office on 2004-06-17 for method and device for collecting and stabilizing a biological sample.
Invention is credited to Augello, Frank A., Bastian, Helge, Oelmuller, Uwe, Rainen, Lynne, Walenciak, Mathew, Wyrich, Ralf.
Application Number | 20040115689 10/610553 |
Document ID | / |
Family ID | 24842996 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040115689 |
Kind Code |
A1 |
Augello, Frank A. ; et
al. |
June 17, 2004 |
Method and device for collecting and stabilizing a biological
sample
Abstract
A collection container and method for collecting a predetermined
volume of a biological sample, and particularly a whole blood
sample, includes an effective amount of at least one stabilizing
agent. The stabilizing agent is able to stabilize nucleic acids in
the biological sample at the point of collection to prevent
enzymatic degradation of the nucleic acids. The stabilizing agents
include cationic compounds, detergents, particularly cationic
detergents, chaotropic salts, ribonuclease inhibitors, chelating
agents, and mixtures thereof.
Inventors: |
Augello, Frank A.; (Cedar
Knolls, NY) ; Rainen, Lynne; (Maplewood, NJ) ;
Walenciak, Mathew; (Madison, NJ) ; Oelmuller,
Uwe; (Erkrath, DE) ; Wyrich, Ralf;
(Grevenbroich, DE) ; Bastian, Helge; (Mettmann,
DE) |
Correspondence
Address: |
ROYLANCE, ABRAMS, BERDO & GOODMAN, L.L.P.
1300 19TH STREET, N.W.
SUITE 600
WASHINGTON,
DC
20036
US
|
Family ID: |
24842996 |
Appl. No.: |
10/610553 |
Filed: |
July 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10610553 |
Jul 2, 2003 |
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09707745 |
Nov 8, 2000 |
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6602718 |
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Current U.S.
Class: |
435/6.16 ;
435/184; 435/287.2 |
Current CPC
Class: |
Y10T 436/10 20150115;
G01N 33/96 20130101; Y10T 436/2525 20150115; Y10T 436/108331
20150115; C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12Q 2527/125
20130101; C12Q 2527/127 20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 435/184 |
International
Class: |
C12Q 001/68; C12N
009/99; C12M 001/34 |
Claims
What is claimed is:
1. An apparatus for collecting a biological sample, said apparatus
comprising: a container defining an internal chamber, and a closure
closing said open end, and at least one stabilizing component
contained within said container in an amount effective to stabilize
and preserve said biological sample.
2. The apparatus of claim 1, wherein said biological sample is
whole blood.
3. The apparaus of claim 1, wherein said stabilizing component is
an aqueous solution of a stabilizing agent selected from the group
consisting of cationic compounds, detergents, chaotropic salts,
ribonuclease inhibitors, chelating agents, and mixtures thereof,
and wherein said aqueous solution of said stabilizing agent has a
pH of about pH 2 to about pH 12.
4. The apparatus of claim 3, wherein said aqueous solution has a pH
of about pH 2 to about pH 10.
5. The apparatus of claim 3, wherein said aqueous solution has a pH
of about pH 3 to about pH 8.
6. The apparatus of claim 1, wherein said stabilizing agent has the
general formula YR.sub.1R.sub.2R.sub.3R.sub.4X wherein Y is
nitrogen or phosphorous; R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of branched alkyl,
non-branched alkyl, C.sub.6-C.sub.20 aryl, and C.sub.6-C.sub.26
aralkyl; and X is an anion.
7. The apparatus of claim 6, wherein X is an anion selected from
the group consisting of phosphate, sulfate, formate, acetate,
propionate, oxalate, malonate, succinate, citrate, bromide and
chloride.
8. The apparatus of claim 6, wherein Y is nitrogen and said
stabilizing agent is a quaternary amine.
9. The apparatus of claim 6, wherein said R.sub.1 is an alkyl
having 12, 14, or 16 carbon atoms and R.sub.2, R.sub.3, and R.sub.4
are methyl.
10. The apparatus of claim 1, wherein said container has an
internal pressure less than atmospheric pressure, for drawing a
predetermined volume of said biological sample into said
container.
11. The apparatus of claim 3, wherein said stabilizing agent is
included in an amount to lyse cells in said biological sample.
12. The apparatus of claim 3, wherein said stabilizing agent lyses
reticulocytes, bacteria, red blood cells, white blood cells, and
viruses.
13. The apparatus of claim 3, wherein said stabilizing agent is
included in an amount to preserve and stabilize nucleic acids in
said biological sample.
14. The apparatus of claim 3, wherein said stabilizing agent is
included in an amount effective to prevent gene induction in said
biological sample.
15. The apparatus of claim 3, wherein said chaotropic salt is
selected from the group consisting of guanidinium isocyanate and
guanidinium hydrochloride.
16. The apparatus of claim 3, wherein said detergent is selected
from the group consisting of sodium dodecylsulfate and
polyoxyethylene sorbitan monolaurate.
17. The apparatus of claim 3, wherein said ribonuclease inhibitor
is placental RNAse inhibitor protein.
18. The apparatus of claim 6, further comprising at least one
proton donor in an effective amount to stabilize nucleic acids in
said sample.
19. The apparatus of claim 18, wherein said portion donor is
selected from the group consisting of alkenyl carboxylic acids,
C.sub.2-C.sub.6 aliphatic mono- and dicarboxylic acids, aliphatic
ketodicarboxylic acids, amino acids, mineral acids and mixtures
thereof.
20. A method of stabilizing a biological sample comprising the
steps of: providing a sample collection container having a side
wall, and a bottom defining an internal chamber, said container
containing at least one stabilizing agent in an amount sufficient
to stabilize and preserve a biological sample; and obtaining a
biological sample and immediately thereafter introducing said
biological sample into said container and mixing said biological
sample with said stabilizing agent to form a stabilized biological
sample.
21. The method of claim 20, wherein said stabilizing agent is in an
aqueous medium containing a component selected from the group
consisting of cationic compounds, detergents, chaotropic salts,
ribonuclease inhibitors, chelating agents, and mixtures
thereof.
22. The method of claim 20, wherein said container has an internal
pressure less than atmospheric pressure, said method comprising
drawing a predetermined volume of said biological sample directly
into said container and mixing with a stabilizing amount of said
stabilizing agent.
23. The method of claim 22, wherein said biological sample is whole
blood and said method comprises withdrawing said whole blood sample
from a patient and introducing said whole blood sample directly
into contact with said stabilizing agent in said sample collection
container to lyse cells and stabilize nucleic acids in said whole
blood sample.
24. The method of claim 20, wherein said biological sample is
selected from the group consisting of red blood cell concentrates,
platelet concentrates, leukocyte concentrates, plasma, serum,
urine, bone marrow aspirates, tissue, and cerebral spinal
fluid.
25. The method of claim 20, wherein said stabilizing agent is
included in an amount to inhibit gene induction in said biological
sample.
26. The method of claim 21, wherein said stabilizing agent is a
cationic compound having the formula the general formula
YR.sub.1R.sub.2R.sub.3R.s- ub.4X wherein Y is nitrogen or
phosphorous; R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of branched alkyl,
non-branched alkyl, C.sub.6-C.sub.20 aryl, and C.sub.6-C.sub.26
aralkyl; and X is an anion.
27. The method of claim 26, wherein X is selected from the group
consisting of phosphate, sulfate, formate, acetate, propionate,
oxalate, malonate, succinate, citrate, bromide and chloride.
28. The method of claim 26, wherein said R.sub.1 is an alkyl having
12, 14, or 16 carbon atoms and R.sub.2, R.sub.3, and R.sub.4 are
methyl.
29. The method of claim 26, wherein Y is nitrogen.
30. The method of claim 21, wherein said aqueous medium has a pH of
about pH 3 to about pH 8.
31. The method of claim 21, wherein said chaotropic salt is
selected from the group consisting of guanidinium isothiocyanate
and guanidinium hydrochloride.
32. The method of claim 21, wherein said detergent is selected from
the group consisting of sodium dodecylsulfate and polyoxyethylene
sorbitan monolaurate.
33. The method of claim 21, wherein said ribonuclease inhibitor is
placental RNAse inhibitor protein.
34. The container of claim 26, further comprising at least one
proton donor in an effective amount to stabilize nucleic acids in
said sample.
35. The container of claim 34, wherein said proton donor is
selected from the group consisting of alkenyl carboxylic acids,
C.sub.2-C.sub.6 aliphatic mono- and dicarboxylic acids, aliphatic
ketodicarboxylic acids, amino acids, mineral acids and mixtures
thereof.
36. A method of collecting and stabilizing a whole blood sample,
said method comprising: providing a sample collection container
having a side wall, a bottom wall and a closure member forming an
internal chamber, said container enclosing an effective amount of
an aqueous solution or dispersion of a nucleic acid stabilizing
agent to stabilize and preserve nucleic acids in a whole blood
sample, said internal chamber having pressure less than atmospheric
pressure; and collecting a whole blood sample directly from a
patient in said collection container and mixing said blood sample
with said stabilizing agent to form a stable whole blood
sample.
37. The method of claim 36, wherein said closure is a septum and
said method comprises piercing said septum with a cannula and
introducing said whole blood sample through said cannula into said
collection container.
38. The method of claim 36, wherein said stabilizing agent is
selected from the group consisting of cationic compounds,
detergents, chaotropic salts, ribonuclease inhibitors, chelating
agents, and mixtures thereof, and wherein said aqueous solution or
dispersion has a pH of about pH 2 to about pH 12.
39. The method of claim 36, wherein said aqueous solution or
dispersion has a pH of about pH 2 to about pH 5.
40. The method of claim 36, wherein said aqueous solution or
dispersion has a pH of about 3.6 to about pH 3.8.
41. The method of claim 36, wherein said stabilizing agent is
selected from the group consisting of guanidinium isothiocyanate,
guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene
sorbitan monolaurate, placental RNAse inhibitor protein, and
mixtures thereof.
42. The method of claim 38, wherein said stabilizing agent has the
general formula YR.sub.1R.sub.2R.sub.3R.sub.4X wherein Y is
nitrogen or phosphorous R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of branched alkyl,
non-branched alkyl, C.sub.6-C.sub.20 aryl, and C.sub.6-C.sub.26
aralkyl; and X is an anion.
43. The container of claim 18, wherein said portion donor is
selected from the group consisting of alkenyl carboxylic acids,
C.sub.2-C.sub.6 aliphatic mono- and dicarboxylic acids, aliphatic
ketodicarboxylic acids, amino acids, mineral acids and mixtures
thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to a method and device for
collecting a biological sample, and particularly a whole blood
sample, directly from a patient. More particularly, the invention
relates to evacuated fluid sample containers having a stabilizing
additive contained therein for stabilizing nucleic acids
immediately on collection of a biological sample.
BACKGROUND OF THE INVENTION
[0002] Sample collection containers have been in common use for
many years for collecting and storing blood and other body fluids
or samples. Typically, the collection containers are glass or
plastic tubes having a resilient stopper. These glass or plastic
tubes are often used for blood collection.
[0003] Blood collection tubes are available where the tube is
evacuated to draw a volume of blood into the tube. The tubes can
have various additives, such as ethylenediaminetetraacetic acid
(EDTA) contained therein for preparing the blood sample for a
particular test. A common additive is an anticoagulation agent.
Typically, the anticoagulation additive is a buffered citrate or
heparin in an aqueous solution. The aqueous citrate is combined
with the blood sample in a specified amount to determine the amount
of an anticoagulant needed for conducting certain tests. These
devices can be used only for serological testing since the
additives do not stabilize the nucleic acids in the sample. During
shipment, labile RNA molecules are degraded enzymatically so that
subsequent RNA separation and analysis is difficult. Furthermore,
mechanical irritation or disruption of cells during blood
collection and transport causes the induction of gene transcription
with the concomitant over- or underproduction of certain mRNA
species.
[0004] Common additives including anticoagulants to maintain the
blood sample in an anticoagulated state are used for performing
various processing steps. For example, anticoagulants are typically
used in blood samples prior to centrifuging to separate the blood
into cell layers. An example of this type of sample tube containing
an anticoagulant is disclosed in U.S. Pat. No. 5,667,963 to Smith
et al.
[0005] In recent years there has been an increase in interest in
the field of biological, medical and pharmacological science in the
study of gene activities and nucleic acids obtained from biological
samples. In particular, ribonucleic acids can provide extensive
information of the genetic origin and functional activity of the
cell. This information may be used in clinical practice to diagnose
infections, detect the presence of cells expressing oncogenes,
detect heredity disorders, monitor the state of host defense
mechanisms and to determine the HLA type or other marker of
identity.
[0006] A number of methods exist for isolating RNA which entails
disruption of the cell and liberating RNA into solution. Other
methods exist for protecting RNA from enzymatic digestion by
endogenous RNases. The RNA can then be separated from the DNA and
protein, which is solubilized along with the RNA. These processes
are usually performed in stepwise fashion rather than for
simultaneously lysing cells, solubilizing RNA and inhibiting
RNases. Some methods for lysing cells and inhibiting RNases are
known that use chaotropic salts of guanidinium.
[0007] A commonly used process for isolating RNA involves
homogenizing cells in guanidinium isothiocyanate, followed by the
sequential addition of sodium acetates and phenol, and
chloroform/isoamyl alcohol. After centrifugation, RNA is
precipitated from the upper layer by the addition of alcohol. Other
methods include the addition of hot phenol to a cell suspension,
followed by alcohol precipitation.
[0008] Anionic and cationic surfactants are used to lyse cells and
liberate cytoplasmic RNA. An example of a method for lysing cells
and simultaneously precipitating RNA and DNA from solution is
disclosed in U.S. Pat. No. 5,010,183 to Macfarlane. In this
process, the RNA is made insoluble. A 2% solution of the surfactant
benzyldimethyl n-hexadecylammonium chloride together with 40% urea
and other additives are added to a cell suspension. The suspension
is then centrifuged to recover a pellet of the insoluble materials.
The pellet is resuspended in ethanol and the RNA and DNA are
precipitated by the addition of a salt.
[0009] A method for analyzing RNA isolated from blood uses
amplification methods including polymerase chain reaction to detect
sequences of RNA in minute amounts. One difficulty in analyzing RNA
is the separation of the RNA from the protein and the DNA in the
cell before the RNA is degraded by nucleases. RNase and other
nucleases are present in the blood in sufficient quantities to
destroy unprotected RNA. Therefore, it is desirable to use a method
of isolating RNA from cells in a manner to prevent hydrolysis of
RNA by nucleases.
[0010] The blood collection methods currently in common use are
able to collect and retain the blood for analysis at a later time.
The collection device can include an anticoagulant to prevent
coagulation during storage. However, the nucleases present in the
blood hydrolyze some RNA species during storage and transport while
mechanical irritation or disruption of cells during blood
collection causes induction of some RNA species. These
preanalytical sample handling factors result in under- or
overrepresentation of mRNA species and eventual degradation of
total RNA as determined by molecular diagnostic test methods. In
addition, gene induction can result in increased levels of RNA in
the sample, which can give false results. Accordingly, there is a
continuing need in the industry for an improved method and
collection device for blood and other biological samples that
preserve the in vivo transcription profile for nucleic acid-based
tests.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to a method and device for
collecting a biological sample. More particularly, the invention is
directed to a collection container and to a method of collecting a
biological sample with a stabilizing additive to stabilize the
sample and preserve the in vivo transcription profile.
[0012] Accordingly, a primary aspect of the invention is to provide
a method and device for collecting a biological sample, and
particularly whole blood, directly from a patient in the presence
of a stabilizer to stabilize and preserve RNA and prevent gene
induction in the sample. The stabilizing additive is present in an
effective amount to stabilize the nucleic acids, particularly RNA,
and stop gene induction.
[0013] A further object of the invention is to provide a method and
device for stabilizing nucleic acids in a biological sample and to
lyse cells, bacteria, viruses and reticulocytes.
[0014] Another object of the invention is to provide a collection
container for receiving and collecting a biological sample where
the container is pre-filled with a measured quantity of a nucleic
acid stabilizing agent.
[0015] A further object of the invention is to provide a method for
stabilizing a biological sample, and particularly whole blood,
immediately upon collection from the patient.
[0016] Still another object of the invention is to provide a method
for preventing induced transcription of RNA in a biological sample
immediately on collection of the biological sample.
[0017] Another object of the invention is to provide an evacuated
container containing an effective amount of a nucleic acid
stabilizing agent, where the container has an internal pressure to
draw a predetermined volume of a biological sample into the
container.
[0018] A further object of the invention is to provide a blood
collection container for collecting an amount of blood and mixing
the blood with a nucleic acid stabilizing agent at the point of
collection to preserve the nucleic acids and prevent gene induction
such that analysis can be conducted at a later time.
[0019] Another object of the invention is to provide a method of
stabilizing blood by collecting the blood sample in a container
having a nucleic acid stabilizing agent and a buffer. The nucleic
acid stabilizing agent can be a detergent, a chaotropic salt, RNase
inhibitors, chelating agents, or mixtures thereof. The pH of the
resulting mixture is adjusted to stabilize the nucleic acids and
promote efficient recovery of the analyte.
[0020] Still another object of the invention is to provide a method
of stabilizing nucleic acids in a blood sample collection device at
about pH 2 to about pH 5 in the presence of at least one
stabilizing agent.
[0021] The objects of the invention are basically attained by
providing an apparatus for collecting a biological sample. The
apparatus includes a container comprising a side wall, a bottom
wall, and an open end defining an internal chamber, and a closure
closing the open end. The container includes at least one
stabilizing component in an effective amount to stabilize and
preserve the biological sample. The container can be pre-filled
with the stabilizing agent.
[0022] The objects of the invention are further attained by
providing a method of stabilizing a biological sample comprising
the steps of: providing a sample collection container having a side
wall, and a bottom defining an internal chamber. The container
contains at least one stabilizing agent in an amount and
concentration sufficient to stabilize and preserve a biological
sample. A biological sample is obtained and immediately introduced
into the container and the biological sample is mixed with the
stabilizing agent to form a stabilized biological sample.
[0023] The objects of the invention are also attained by providing
a method of collecting and stabilizing a whole blood or other
biological sample. The method comprises providing a sample
collection container having a side wall, a bottom wall and a
closure member forming an internal chamber. The container encloses
an effective amount of an aqueous solution or dispersion of a
nucleic acid stabilizing agent to stabilize and preserve nucleic
acids and/or the transcriptional profile in a whole blood sample.
The internal chamber has pressure less than atmospheric pressure. A
whole blood sample is collected directly from a patient in the
collection container and the blood sample is mixed with the
stabilizing agent to form a stable whole blood sample.
[0024] These objects, advantages and other salient features of the
invention will become apparent from the annexed drawing and the
following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWING
[0025] The following is a brief description of the drawing, in
which:
[0026] FIG. 1 is a cross-sectional side view of the container in
one embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention is directed to a method and device for
stabilizing and preserving a biological sample. More particularly,
the invention is directed to a method and device for stabilizing
nucleic acids and/or preventing gene induction in a biological
sample. In preferred embodiments of the invention, the device is a
pre-filled container containing an amount of a nucleic acid
stabilizing agent.
[0028] The biological sample can be a body fluid withdrawn from a
patient. In one embodiment, the biological fluid is whole blood.
Examples of other biological samples include cell-containing
compositions such as red blood cell concentrates, platelet
concentrates, leukocyte concentrates, plasma, serum, urine, bone
marrow aspirates, cerebral spinal fluid, tissue, cells, and other
body fluids.
[0029] In a preferred embodiment, the device 10 is for drawing a
blood sample directly from a patient for stabilizing the nucleic
acids immediately at the point of collection. Referring to FIG. 1,
device 10 includes a container 12 defining a chamber 14. In the
embodiment illustrated, container 12 is a hollow tube having a side
wall 16, a closed bottom end 18 and an open top end 20. Container
12 is dimensioned for collecting a suitable volume of a biological
fluid. A resilient closure 22 is positioned in open top end 20 to
close container 12. Preferably, closure 22 forms a seal capable of
effectively closing container 12 and retaining a biological sample
in chamber 14. A protective shield 23 overlies closure 22.
[0030] Container 12 can be made of glass, plastic or other suitable
materials. Plastic materials can be oxygen impermeable materials or
contain an oxygen impermeable layer. Alternatively, container 12
can be made of a water and air permeable plastic material.
Preferably, chamber 14 maintains a pressure differential between
atmospheric pressure and is at a pressure less than atmospheric
pressure. The pressure in chamber 14 is selected to draw a
predetermined volume of a biological sample into chamber 14.
Typically, a biological sample is drawn into chamber, 14 by
piercing closure 22 with a needle 24 or cannula as known in the
art. An example of a suitable container 12 and closure 22 are
disclosed in U.S. Pat. No. 5,860,937 to Cohen, which is hereby
incorporated by reference in its entirety.
[0031] Container 12 is preferably made of a transparent material.
Examples of suitable transparent thermoplastic materials include
polycarbonates, polyethylene, polypropylene, polyethylene-terephtha
late. Container 12 has a suitable dimension selected according to
the required volume of the biological sample being collected. In
one embodiment, container 12 has a tubular shape with an axial
length of about 100-mm and a diameter of about 13-mm to 16-mm.
[0032] Closure 22 is made of a resilient material capable of
maintaining an internal pressure differential less than atmospheric
and that can be pierced by a needle to introduce a biological
sample into container 12. Suitable materials for closure include,
for example, silicone rubber, natural rubber, styrene butadiene
rubber, ethylene-propylene copolymers and polychloroprene.
[0033] Container 12 also contains a stabilizing additive 26. The
stabilizing additive 26 is preferably a liquid containing a
stabilizing agent and is included in an effective amount to mix
with the biological sample and stabilize the nucleic acids and/or
prevent gene induction of the cells or nucleic acids contained
therein. In one embodiment, the internal pressure of container 12
and the volume of stabilizing additive 26 are selected to provide
the necessary concentration of the stabilizing agent for the volume
of the biological sample collected. In one preferred embodiment,
the internal pressure of container 12 is selected to draw a
predetermined volume of about 2.5 ml of a biological sample into
container 12 containing an effective volume of stabilizing additive
26 for stabilizing the volume of the biological sample. In
alternative embodiments, container 12 can have an internal pressure
at substantially atmospheric pressure.
[0034] In one embodiment, container 12 is made of a plastic that is
water and gas permeable. Water loss by evaporation of the
stabilizing agent through the permeable wall of the container
increases the concentration of the stabilizing agent and decreases
the pressure within the container. The diffusion of oxygen through
the wall of the tube has the effect of decreasing the vacuum in the
container. The water and oxygen permeability properties of the
container are selected to maintain the desired pressure
differential within the container for the desired shelf life of the
container. The shelf life is optimized by balancing the oxygen
permeability with the water loss. Preferably, the container has a
shelf life of about one year.
[0035] Stabilizing additive 26 is typically an aqueous solution or
dispersion of at least one active stabilizing agent that is
included in the container as a pre-filled container. Stabilizing
additive 26 preferably contains at least one stabilizing agent in a
concentration capable of stabilizing nucleic acids in the
biological sample, and particularly a whole blood sample. The
stabilizing agents are preferably able to stabilize effectively DNA
and RNA including mRNA, tRNA and cRNA. Examples of suitable
stabilizing agents for stabilizing and preserving nucleic acids
and/or preventing gene induction include cationic compounds,
detergents, chaotropic salts, ribonuclease inhibitors, chelating
agents, and mixtures thereof. A suitable ribonuclease inhibitor is
placental RNAse inhibitor protein. Examples of chaotropic salts
include urea, formaldehyde, guanidinium isothiocyanate, guanidinium
hydrochloride, formamide, dimethylsulfoxide, ethylene glycol and
tetrafluoroacetate.
[0036] The stabilizing agent can also include another component for
treating the biological sample. For example, chemical agents can be
included to permeabilize or lyse viruses and cells. Other
components include proteinases, phenol, phenol/chloroform mixtures,
alcohols, aldehydes, ketones and organic acids.
[0037] The detergents can be anionic detergents, cationic
detergents or nonionic detergents. The anionic detergent can be,
for example, sodium dodecyl sulfate. Nonionic detergents can be,
for example, ethylene oxide condensation products, such as
ethoxylated fatty acid esters of polyhydric alcohols. A preferred
nonionic detergent is a polyoxyethylene sorbitan monolaurate sold
under the trade name TWEEN 20 by Sigma Chemical Co. The detergents
are included in an effective amount to lyse the cells and form
micelles and other complexes with the nucleic acids.
[0038] In preferred embodiments, the stabilizing agent is a
cationic compound having the general formula
YR.sub.1R.sub.2R.sub.3R.sub.4 X wherein Y is nitrogen or
phosphorous; R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently branched or non-branched alkyl, C.sub.6-C.sub.20
aryl, or C.sub.6-C.sub.26 aralkyl, and X is an organic or inorganic
anion.
[0039] The anion can be an anion of an inorganic acid such as the
HX where X is fluorine, chlorine, bromine or iodine, with chlorine
and bromine being preferred. The anion can also be the anion of a
mono-, di- or tricarboxylic acid. Typically, the anion of the
cationic compound is selected from the group consisting of
phosphate, oxalate, malonate, succinate, citrate, bromide and
chloride.
[0040] When R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are aryl groups,
the aryl groups independently can be, for example, phenyl, lower
alkyl-substituted benzyl, and/or halogenated benzyl. In one
embodiment R.sub.1 is a C.sub.12, C.sub.14, or C.sub.16 alkyl and
R.sub.2, R.sub.3, and R.sub.4 are methyl groups. In a preferred
embodiment, Y is nitrogen and the stabilizing agent is a quaternary
amine. Suitable quaternary amines include alkyltrimethylammonium
where the alkyl group has 12, 14 or 16 carbons. One preferred
cationic compound is tetradecyltrimethyl ammonium oxalate. Other
suitable quaternary amines include alkyltrimethylammonium where the
alkyl group includes 12, 14, 16 or 18 carbons. Examples of suitable
quaternary amine surfactants are disclosed in U.S. Pat. No.
5,728,822 to Macfarlane, which is hereby incorporated by reference
in its entirety.
[0041] In preferred embodiments of the invention the stabilizing
agent is a cationic compound and includes a proton donor. It has
been found that the addition of a proton donor to the cationic
compounds increases the ability of the cationic compounds to
stabilize the nucleic acids in the biological sample. Examples of
suitable proton donors include monocarboxylic acids, alkenyl
carboxylic acids, C.sub.2-C.sub.6 aliphatic mono- and dicarboxylic
acids, aliphatic ketodicarboxylic acids, amino acids, mineral acids
and mixtures thereof. Examples of suitable aliphatic carboxylic
acids include C.sub.1-C.sub.6 alkyl carboxylic acids, such as
acetic acid, propionic acid, n-butanoic acid, n-pentanoic acid,
isopentanoic acid, 2-methylbutanoic acid, 2,2 dimethylpropionic
acid, n-hexanoic acid, n-octanoic acid, n-decanoic acid, and
dodecanoic acid. Examples of alkenyl carboxylic acids include
acrylic acid, methacrylic acid, butenoic acid, isobutenoic acid and
mixtures thereof.
[0042] The dicarboxylic acids of the proton donor are selected from
the consisting of oxalic acid, malonic acid, succinic acid,
glutaric acid, adipic acid and mixtures thereof. Examples of
hydroxyl-containing acids include tartaric acid and malic acid.
Suitable amino acids are selected from the group consisting of
glycine, alanine, valine, leucine, iso-leucine and mixtures
thereof.
[0043] The quantity of stabilizing additive 26 in container 12 is
determined by the internal volume of container 12, the internal
pressure and the volume of the biological sample drawn into the
container. In the illustrated embodiment, container 12 has an axial
length of about 100-mm and a diameter of about 16-mm and has an
internal pressure to draw a biological sample of about 2.5-ml.
Stabilizing additive 26 typically contains about 50 mg to about 90
mg per ml of the carrier liquid. Preferably, stabilizing additive
26 is an aqueous medium containing about 60 mg/ml to about 80
mg/ml, and most preferably about 70 mg/ml. The volume of
stabilizing additive 26 in container 12 is about 6 to 8 ml, and
preferably about 7 ml.
[0044] In one preferred embodiment, stabilizing additive 26
includes about 70 mg/ml of a nucleic acid stabilizing agent and is
mixed with whole blood drawn directly from a patient. The blood is
mixed with the liquid in a ratio of about 1:2 to about 1:3.5,
preferably about 1:2.5 to about 1:1.3, and most preferably about
1:2.7 to about 1:2.8 by volume.
[0045] The concentration of the stabilizing agent is sufficient to
stabilize the nucleic acids. In one preferred embodiment, the
biological sample is whole blood. The concentration of the
stabilizing agent in an amount of about 8 to about 12 mg/ml of
blood, preferably about 9 to about 11 mg/ml, and more preferably
about 10 mg/ml of blood.
[0046] The method of the invention is performed by obtaining a
biological sample and introducing the sample into the container
containing the stabilizing additive. In preferred embodiments the
biological sample is prepared and immediately introduced directly
into the collection container. In certain embodiments, the
biological sample is introduced directly into the collection
container with out any intervening process steps. It has been found
that collecting the biological sample directly from the patient,
such as when collecting a whole blood sample, and introducing the
sample directly into the container substantially prevents or
reduces the decomposition of the nucleic acids that otherwise occur
when the sample is stored before combining with the stabilizing
agent. In addition, is has been found that the introduction of the
biological sample immediately upon collection or preparation
reduces or prevents gene induction.
[0047] The cationic compounds are preferred stabilizing agents
since the cationic group is able to effectively attack the
negatively charged groups of the nucleic acids. The cationic groups
of the compound for non-covalent bonds with the negatively charged
phosphate groups of the nucleic acid backbone. The hydrophilic, and
particularly the aliphatic, portion of the detergent molecules form
precipitatable micelles and complexes containing the nucleic acids.
The resulting micelles isolate the nucleic acids from the
endogenous ribonucleases to prevent enzymatic degradation. The
cationic compounds produce a stabilized whole blood sample that can
be transported at ambient temperature to a laboratory where the
nucleic acids can be isolated from the sample.
[0048] It has been found that the recovery and stabilization of
nucleic acids in the biological sample is dependent on the pH of
the biological sample and stabilizing agent. The pH of the
resulting mixture can range from about pH 2 to about pH 12,
preferably about pH 2 to about pH 10, and more preferably about pH
3 to about pH 8. The life of the nucleic acids in this range will
vary depending on the biological sample, the ratio of the amount of
the biological sample to the amount of the stabilizing agent, and
the particular stabilizing agent used. The shelf life of the
stabilized nucleic acids in this pH can range from about 24 hours
to several days.
[0049] The pH of the resulting mixture will vary depending on the
biological sample being stabilized. In one embodiment of the
invention, the biological sample is whole blood and the mixture of
the whole blood and the stabilizing agent is adjust to about pH 2
to about pH 5. Nucleic acids stabilized with cationic compounds
adjusted to about pH 2 to about pH 5 are stable at ambient
temperature for several days. It has been found that optimum long
term stabilization of nucleic acids in whole blood is obtained at
about pH 3.6 to about pH 3.8. In other biological samples, the pH
is adjusted to stabilize the mixture. For example, it has been
found that eucaryotic cell cultures and bacteria are stabilized at
pH 4 to about pH 8, and preferably at about pH 6 to about pH 8.
[0050] The pH of the mixture of the biological sample and
stabilizing agent can be adjusted by the addition of a suitable
buffer. An example of a buffer that has been found to be effective
in adjusting the pH of the biological sample is tartaric acid.
Other buffers and pH adjusting agents as known in the art can also
be used. The pH of the buffer can be adjusted to the desired range
by the addition: of sodium hydroxide.
[0051] The nucleic acids, either DNA or RNA can be separated from
the stabilized biological sample using various processes as known
in the art. It has been found that the stabilizing agents can be
separated from the nucleic acids during the purification protocol
performed in the laboratory to yield the purified nucleic acid.
[0052] Cationic compounds cause lysis of the cells and virus in the
sample and precipitation of the nucleic acids in a complex with the
compound. The precipitated nucleic acids can be extracted from the
complex by a phenol extraction or by a formamide buffer as known in
the art. In a further embodiment, the detergent can be solubilized
to disassociate the complex and leave the insoluble nucleic acids.
The compound can be solubilized by treating the complex with a
concentrated solution of lithium chloride. Other methods of
isolating and purifying nucleic acids are disclosed in U.S. Pat.
No. 5,990,301 to Colpan et al., which is hereby incorporated by
reference in its entirety.
EXAMPLE 1
Stabilization of RNA in Human Blood
[0053] This example demonstrates the effects of the ratio of the
blood to stabilizing agent and the concentration of the stabilizing
agent.
[0054] Twenty-four samples were prepared for this comparison. Each
sample was prepared from 2.5 ml blood, drawn with a sodium citrate
containing blood collection device, and mixed with 7.5 ml of a
stabilization buffer containing 3% (w/v)
tetradecyltrimethylammonium oxalate and 125 mM and 200 mM tartaric
acid, respectively, in a 12 ml polyethylene tube. The pH of the
buffer was adjusted with sodium hydroxide to 3.3, 3.5 and 3.7,
respectively. Samples were stored at room temperature for 25 hours
and 72 hours, respectively. In order to isolate the cellular RNA,
the tubes were centrifuged at 5000.times.g for 10 minutes. The
supernatant was discarded and the pellet was washed once with
water. After additional centrifugation at 5000.times.g for 10
minutes, the pellet was dissolved in 300 .mu.l of a lysis buffer,
i.e., buffer RLT (QIAGEN GmbH), diluted with 360 .mu.l water and 40
.mu.l proteinase K were added. After a proteinase digestion for 10
minutes at 55.degree. C. the sample was centrifuged at
20,000.times.g for 3 minutes, the supernatant was transferred into
a new tube and 350 .mu.l of 98% ethanol were added. The sample was
then applied to a silica membrane containing spin column via
centrifugation at 8000.times.g for 1 minute. The spin column was
washed once with a GITC containing washing buffer-like buffer RW1
(QIAGEN GmbH) and two times with a ethanol containing buffer-like
buffer RPE (QIAGEN GmbH). The RNA was then eluted from the silica
membrane with 2.times.40 .mu.l of RNase free water. All samples
were processed in duplicates.
[0055] The yield of the isolated RNA was determined by measuring
the optical density at 260 nm wavelength in a spectrophotometer and
calculating that 1 OD260 corresponds to a concentration of 40 .mu.g
RNA/ml. The integrity of the isolated RNA was proved by
electrophoresis of 30 .mu.l of the eluate in a denaturating
agarose/formaldehyde gel, stained with ethidium bromide. The yield
of the RNA is presented in Table 1 and Table 2.
1TABLE 1 125 mM Tartaric Acid Sample pH Storage Time (hours) Yield
(.mu.g) 1 3.3 24 8.4 2 3.3 24 7.6 3 3.5 24 9.5 4 3.5 24 9.8 5 3.7
24 13.3 6 3.7 24 17.2 7 3.3 72 7.2 8 3.3 72 6.8 9 3.5 72 10.3 10
3.5 72 10.9 11 3.7 72 14.8 12 3.7 72 16.1
[0056]
2TABLE 2 200 mM Tartaric Acid Sample pH Storage Time (hours) Yield
(.mu.g) 13 3.3 24 5.9 14 3.3 24 7.4 15 3.5 24 10.6 16 3.5 24 10.9
17 3.7 24 17.2 18 3.7 24 18.5 19 3.3 72 5.1 20 3.3 72 5.3 21 3.5 72
7.2 22 3.5 72 7.1 23 3.7 72 13.3 24 3.7 72 16.6
[0057] the results show that for the blood volume of 2.5 ml mixed
with 7.5 ml of stabilization buffer containing 3% (w/v)
tetradecyltrimethylammoniu- m oxalate and 125 mM or 200 mM tartaric
acid, respectively, the pH of 3.7 is optimal for the yield and
integrity of the total RNA. With all pH values, the stabilization
of the RNA, judged by the integrity of the ribosomal RNA, was very
good, but the yield of the isolated RNA was lower with the buffers
adjusted to pH 3.3 and 3.5, respectively, than with the buffer
adjusted to pH 3.7. However, even the lower yields achieved with
the stabilization buffer adjusted to pH 3.3 were comparable or
slightly better than the yields achieved with a control method, the
RNA isolation with the QIAamp.RTM. RNA Blood Mini Kit (QIAGEN Cat.
No. 52303), which showed an average yield of 6.8 .mu.g RNA per 2.5
ml of blood.
EXAMPLE 2
Northern-Blot Anaylsis
[0058] This example shows the results of a Northern-Blot analysis
performed with blood samples from three different donors stored at
room temperature for 1 hour, 24 hours, 48 hours and 72 hours.
[0059] 2.5 ml blood samples, drawn with a sodium citrate containing
blood collection device, were mixed with 6.9 ml of stabilization
buffer containing 4% (w/v) tetradecyltrimethylammonium oxalate and
200 mM tartaric acid in a 16.times.100 mm polyethylene tube.
Samples were stored at room temperature for 1 hour, 24 hours 48
hours and 72 hours, respectively. In order to isolate the cellular
RNA, the tubes were centrifuged at 5000.times.g for 10 minutes. The
supernatant was discarded and the pellet was washed once with
water. After additional centrifugation at 5000.times.g for 10
minutes, the pellet was dissolved in 300 .mu.l of a lysis buffer,
i.e., buffer RLT (QIAGEN GmbH), diluted with 360 .mu.l water and 40
.mu.l proteinase K were added. After a proteinase digestion for 10
minutes at 55.degree. C. the sample was centrifuged at
20,000.times.g for 3 minutes, the supernatant was transferred into
a new tube and 350 .mu.l of 98% ethanol were added.
[0060] The sample was then applied to a silica membrane containing
spin column by centrifugation at 8000.times.g for 1 minute. The
spin column was washed once with a GITC containing washing
buffer-like buffer RW1 (QIAGEN GmbH) and two times with a ethanol
containing buffer-like buffer RPE (QIAGEN GmbH). The RNA was then
eluted from the silica membrane with 2.times.40 .mu.l of RNase free
water. A single sample was prepared for each variable. 2.5 .mu.g of
the isolated RNA were loaded onto a denaturating
agarose/formaldehyde gel, and after the electrophoresis the RNA was
transferred onto a nylon membrane. The nylon membrane was
hybridized subsequently with a radioactive labeled RNA probe, which
contained the sequence of an IFN-gamma inducible gene (GeneBank
Acc.No. L07633) overnight at 60.degree. C., washed several times at
60.degree. C. with washing buffers containing 2.times.SSC/0.1% SDS
to 0.5.times.SSC/0.1% SDS. The nylon membrane was exposed
subsequently to an X-ray film. As a control, RNA from the same
donor was isolated using TRIzol.TM. LS reagent (Life Technologies)
directly after the blood draw and analyzed as described above.
[0061] The results show that the transcript levels of the IFN-gamma
inducible gene, which was used as a probe to hybridize the isolated
RNA, was preserved over the entire time period with no visible
change in the expression level. The transcript levels were equal to
the TRIzol.TM.LS controls. These controls represent the in vivo
conditions of the sample at the time point of the blood draw
because the TRIzol reagent contains phenol combined with guanidine
isothiocyanate and is considered as a reagent that destroys cells
immediately, denatures proteins and therefore completely inhibits
any biological activity. The comparison of the signal intensities
from the stored samples with the TRIzol controls in the
Northern-Blot analysis indicates that the transcript levels of the
IFN-gamma inducible gene were "frozen" immediately after addition
of the stabilization buffer to the blood sample and did not change
any more during storage.
EXAMPLE 3
Comparison of Blood Collection Device with Conventional EDTA
Tube
[0062] This example compares the stabilization of RNA with the
collection device of the present invention and conventional EDTA
containing tube.
[0063] 2.5 ml blood, drawn from one donor with a blood collection
device, containing 6.9 ml of stabilization buffer (4% (w/v)
tetradecyltrimethylammonium oxalate, 200 mM tartaric acid, pH 3.7)
in a 16.times.100 mm polyethylene tube closed with a HEMOGARD.TM.
closure (Becton, Dickinson and Company) and evacuated to a defined
vacuum that drawed 2.5 ml of blood when connected to the vein of
the donor. Samples were stored at room temperature for 1 hour, 1
day, 3 days, 7 days and 10 days, respectively. In order to isolate
the cellular RNA, the tubes were centrifuged at 5000.times.g for 10
min. The supernatant was discarded and the pellet was washed once
with water. After additional centrifugation at 5000.times.g for 10
min, the pellet was dissolved in 360 .mu.l of a resuspension buffer
containing ammonium acetate and then 300 .mu.l of a lysis buffer,
i.e., buffer RLT (QIAGEN GmbH), and 40 .mu.l proteinase K were
added. After a proteinase digestion for 10 minutes at 55.degree.
C., the sample was centrifuged at 20,000.times.g for 3 minutes, the
supernatant was transferred into a new tube and 350 .mu.l of 98%
ethanol were added. The sample was then applied to a silica
membrane containing spin column by centrifugation at 8000.times.g
for 1 minute. The spin column was washed once with a GITC
containing washing buffer-like buffer RW1 (QIAGEN GmbH) and two
times with a ethanol containing buffer-like buffer RPE (QIAGEN
GmbH).
[0064] A digestion of the residual genomic DNA which could be
co-purified with the RNA in low amounts was performed on the silica
membrane according to the instructions in the manual of the
RNase-Free DNase Set (QIAGEN GmbH Cat.No. 79254). The RNA was
eluted from the silica membrane with 2.times.40 .mu.l of elution
buffer. All samples were processed in duplicates. For the analysis,
the eluates were diluted 1:125 fold and 1 .mu.l of the diluted
eluate was analyzed by real time TaqMan RT-PCR. The mRNA of the
GAPDH-gene was amplified using an assay developed by Perkin Elmer.
Each sample was analyzed in duplicate in the TaqMan RT-PCR
amplification.
[0065] As a control, RNA from the same donor was drawn with a
Becton Dickinson Vacutainer EDTA tube and was stored in this tube
for the same time period as described above. The RNA from 1 ml of
the stored blood sample was isolated at each time point using
TRIzol.TM.LS reagent (Life Technologies). The isolated RNA was
subsequently cleaned up according to the RNeasy.TM. Mini protocol
for the RNA clean up (QIAGEN Cat.No. 74103). The RNA was eluted
with 2.times.40 .mu.l of RNase-free water. The eluate was diluted
1:50 fold in order to compensate for the lower sample volume
processed with the TRIzol method, compared to the 2,5 ml of blood
in the sample tubes. The samples were analyzed using also the GAPDH
TaqMan RT-PCR system from Perkin Elmer.
[0066] The real time RT-PCR results show that in the unpreserved
EDTA blood, the transcript level decreases over time (indicated by
the increasing ct value in the TaqMan analysis) up to a degree of
degradation after 7 to 10 days at which point the mRNA is no longer
detectable. On the other hand, the GAPDH mRNA in the preserved
samples does not show any decrease in copy number, taking into
consideration that the error range of the TaqMan assay is .+-.1 ct
value. Within this error range, all changes in the ct value have to
be considered as normal fluctuations of the amplification system
and no degradation is visible. This result clearly indicates the
advantage of the new developed blood collection device over the
EDTA blood collection tube and also makes clear that the
stabilization of the RNA is a prerequisite for the molecular
analysis of the sample material. It was also possible to isolate
the genomic DNA from the stabilized blood sample. Table 3 shows the
results for the stabilization of genomic DNA in human blood. After
24 hour and 72 hours of storage at room temperature, isolated
genomic DNA was of high molecular weight. The main band migrated at
a length greater than 20 kb. The yield was in the range between 47
to 80 .mu.g per 2.5 ml of blood, which is within the expected yield
range for this amount of blood. The DNA was also applicable to
enzymatic reactions like restriction endonuclease digestion and PCR
amplification.
3TABLE 3 storage at NA stabilization EDTA tube/ mean room temp.
device/ct value mean value/ct ct value value/ct 1 h 33.38 30.17
31.42 29.63 31.48 30.58 31.06 32.29 30.06 30.24 1 day 31.28 30.18
28.62 29.35 30.11 31.26 30.34 33.2 30.19 32.32 3 days 31.27 33.33
31.92 32.37 30.91 36.32 30.15 40 30.3 39.58 7 days 33.03 40 31.16
39.01 32.58 38.4 34.21 37.67 31.9 36.12 10 days 34.2 40 32.47 40
32.58 38.97 32.36 38.38
EXAMPLE 4
Stabilization of Genomic DNA in Whole Blood
[0067] 2.5 ml blood, drawn with a sodium citrate containing blood
collection device, were mixed with 6.9 ml of stabilization buffer
containing 4% (w/v) tetradecyltrimethylammonium oxalate and 200 mM
tartaric acid in a 16.times.100 mm polyethylene tube. Samples were
stored at room temperature for 24 hours and 72 hours, respectively.
In order to isolate the genomic DNA, the tubes were centrifuged at
5000.times.g for 10 min. The supernatant was discarded and the
pellet was washed once with water. After additional centrifugation
at 5000.times.g for 10 minutes, the pellet was dissolved in 300
.mu.l of a EDTA and sodium chloride containing buffer and 400 .mu.l
of a lysis buffer, i.e., buffer AL (QIAGEN GmbH), and 20 .mu.l
proteinase K were added. After a proteinase digestion for 10
minutes at 65.degree. C., 420 .mu.l of 98% ethanol were added. The
sample was then applied to a silica membrane containing spin column
by centrifugation at 8000.times.g for 1 minute. The spin column was
washed once with a guanidine hydrochloride containing washing
buffer-like buffer AW1 (QIAGEN GmbH) and once with an ethanol
containing buffer-like buffer AW2 (QIAGEN GmbH). The DNA was then
eluted from the silica membrane with 300 .mu.l of a
tris-buffer.
[0068] 5 .mu.l of the eluate was analyzed on a 0.8% agarose/TBE gel
stained with ethidium bromide. The yield of the isolated DNA was
determined by measuring the optical density at 260 nm wavelength in
a spectrophotometer and calculating that 1 OD260 corresponds to a
concentration of 50 .mu.g DNA/ml.
[0069] The genomic DNA was also applied to enzymatic reactions like
restriction enzyme digestion or PCR amplification. For the
restriction endonuclease digestion, 2 .mu.g of the DNA was digested
with 6 U EcoRI (E) and Hind III (H), respectively, for 3 hours at
37.degree. C. and analyzed subsequently on a 0.8 % agarose TBE gel.
For the PCR amplification, 150 and 300 ng of the DNA were added to
a 50 .mu.l total volume PCR reaction mix and a 1.1 kb fragment of
the human homologue of giant larvae-gene was amplified. The PCR
products were analyzed on a 1.2-% agarose/TBE gel.
[0070] While various embodiments have been chosen to demonstrate
the invention, it will be understood by those skilled in the art
that various modifications and additions can be made without
departing from the scope of the invention as defined in the
appended claims.
* * * * *