Indian Patents. 207201:"VESSEL FOR WITHDRAWING BLOOD"

Title of Invention	"VESSEL FOR WITHDRAWING BLOOD"
Abstract	The present invention relates to a vessel for withdrawing blood, the vessel containing a solution which comprises a guanidinium salt, a buffer substance, a reducing agent, and/or a detergent as components. The vessel is particularly well suited to sampling blood which is to be analyzed with respect to nucleic acids.

Full Text	VESSEL FOR WITHDRAWING BLOOD The present invention relates to a vessel for withdrawing blood, the blood withdrawn being specifically intended for use in stabilizing and analyzing nucleic acids. When blood is taken, it is normally collected in vessels which already contain anticoagulants such as heoarin. citrate or EDTA. The blood is thereby prevented from coagulating. The blood samples obtained thereby can be stored at suitable temperatures for a long time. This way of obtaining blood has, however, considerable drawbacks if nucleic acids such as (m)RNA or DNA are to be analyzed. For such purposes the nucleic acids contained in the sample should optimally be stabilized immediately at the time of withdrawal, i.e. a degradation of the nucleic acids present should be prevented, but also the new synthesis of mRNA. This objective of a stable storage of the nucleic acids contained in the sample material, i.e. from the moment of withdrawal, has not been achieved yet in practice for the following reasons: Cells contain nucleases, enzymes, which destroy nucleic acids as soon as they come into contact with the substrates thereof (RNA, DNA). The effect of cellular and extracellular nucleases is normally under physiological control as long as the cells are in their normal environment. The withdrawal of blood causes more or less significant changes in the nucteiGacMsmDMngd inihe^alls. Nucleases are then released within the cells and/or by the lysis of cells to the outside. Moreover, nucleic acids are synthetized to a greater or lesser degree. In particular the lona-term storage of blood leads to aaina and destruction of the cells !A 2 Another problem arising with long-term storage of blood samples obtained using standard withdrawal methods is the considerable change in the sample material. Such changes, e.g. strong lysis of cells, may have the effect of preventing the standard methods for isolating nucleic acids from functioning in a sufficiently efficient manner. Apart from the problems relating to stable storage of nucleic acids contained in the sample material, further difficulties arise in the conventional method for withdrawing blood. Conventional anticoagulants are often not separated efficiently enough when isolating nucleic acids and interfere with the subsequent analysis of nucleic acids, e.g. during amplification by means of PCR (polymerase chain reaction). Heparin is e.g. a generally known inhibitor of PCR. Finally, another issue in the quantitative analysis of nucleic acids is how the method as a whole from sampling through to measuring the nucleic acids can be controlled under standardized conditions. Ideally, a quantitatively and qualitatively defined standard nucleic acid should be added to the sample material during the withdrawal stage and should be subjected to the whole process of sampling and determination. This can also not be accomplished with conventional withdrawal systems. A further drawback of conventional blood withdrawal is the risk of transferring infectious material because manual process steps have so far been needed in order to isolate the nucleic acids. Contact with potentially infectious germs cannot be ruled out. The background literature describes a method in which the blood sample is mixed with guanidinium salt directly after withdrawal from a patient (EP 0 818 542 A1). In this method the guanidinium salt is present in powder form in order to take advantage of the greater stability of the guanidinium salt. This method, however, 3 has serious drawbacks because, for instance, the salt must first dissolve in the added blood. This dissolution process depends, in particular, on the temperature and cannot be controlled because of the nontransparent sample material used. The use of this type of product for diagnostic medical purposes is thus very problematic. Furthermore, nucleases are extremely active enzymes which can only be inhibited under extremely denaturing conditions. Denaturation depends on the concentration of the guanidinium salt in solution. The cited method does not specify in any way that the concentration of guanidinium salt in solution should be of a concentration that will have an inhibiting effect. Thus, there is an uncontrolled degradation of nucleic acids during the dissolution process. Moreover, in this method the addition of reducing agents is omitted, without which effective inhibition, in particular of RNases, can not be guaranteed (see Example no. 5). Moreover, the sample prepared in this way can not directly be used for subsequent isolation of the nucleic acid on glass surfaces. Furthermore, the use of guanidinium salt powder does not permit the addition of internal nucleic acid standards. Such standards are mandatory for process control and exact quantification. The present invention was based on the technical problem of providing a vessel for withdrawing blood which does not have the drawbacks of the prior art. In particular, it should be possible to subject the sample taken with the vessel directly to the standard methods for analyzing nucleic acids without the need for further sample preparation steps. In accordance with the invention, this problem is solved by a vessel for withdrawing blood, the vessel containing an aqueous solution comprising the following components: 4 a guanidinium salt; a buffer substance; a reducing agent; and/or a detergent. The vessel of the invention has the following advantages: 1. Blood is already lysed at the moment of withdrawal in that the withdrawal vessel already contains a nucleic acid-stabilizing substance in solution. 2. The nucleic acid-stabilizing substance is composed such that the sample material, in particular the nucleic acids contained therein, are immediately stabilized upon contact with the solution. 3. In contrast to all of the former standard withdrawal systems, such as EDTA or heparin-containing withdrawal vessels, the stabilized sample need no longer be handled as infectious material. 4. The nucleic acid-stabilizing substance is composed such that the sample material can be used directly in subsequent isolating methods. 5. The nucleic acid-stabilizing substance can be separated during subsequent isolation so efficiently that no inhibition of PCR is observed. 6. An internal standard may be added to the nucleic acid-stabilizing substance. This allows the whole method to be controlled, from the moment of sampling up to the detection of nucleic acids. The withdrawal vessel mentioned under item 1 is a conventional blood withdrawing vessel (small tube) into which a defined volume of a nucleic acid-stabilizing substance is introduced. A small tube is then preferably placed under a defined vacuum which guarantees that only a specific volume of blood can flow thereinto during withdrawal. The small tube can be handled by conventional blood-taking methods. The solution contained in the tube contains the following reagents in a specially preferred embodiment: Guanidinium thiocyanate, Triton-X-100, dithiothreitol and a suitable buffer system, such as citrate, Tris or HEPES. In the described composition the solution is compatible with the vacuum tube. This solution can be stored in the vacuum tube without any problems and without any 5 impairment to the desired stabilizing function. The whole system presents no problems, in particular to blood donors, and is safe during sampling. The solution containing the guanidinium salt, the buffer substance, the reducing agent and/or the detergent is stable in storage and converts the supplied and freshly taken blood into a material which is also stable in storage and can be transferred directly to standard nucleic-acid analysis kits (e.g. those of Roche or Qiagen). Guanidinium thiocyanate and/or guanidinium chloride are preferred as the guanidinium salt. Preferably, the guanidinium salt is present in a concentration of 2.0 to 8.0 M. Tris or citrate is preferred as the buffer substance, the exact pH being preferably adjusted with HCI. Other possible buffers are however HEPES, MOPS, citrate and phosphate buffer, such as PBS. The buffer concentration is preferably between 10 and 300 mM, particularly preferably between 10 and 100 mM. Triton-X-100 is preferred as the detergent. Other possible detergents are NP-40, Tween 20, polydocanol or other detergents. The detergent concentration is preferably 5 to 30% (w/v), particularly preferably 10 to 20% (w/v). DTT is preferred as the reducing agent, but S-mercaptoethanol, TCEP (Tris(2-carboxyethyl)phosphine) or other reducing agents can also be used. 6 The preferred concentration of the reducing agent is 0.1 to 10% (w/v), particularly preferred are 0.5 to 2% (w/v). The pH of the solution is preferably 3.0 to 9.0, particularly preferably 4.0 to 7.5, particularly preferably 5 to 6. The pH of the solution is chosen in particular such that a pH ranging from 5.0 to 7.6 is set after addition of the sample material. Particularly preferred is a pH between 6.3 and 6.9 (see Example no. 8). A particularly preferred solution preferably contains 4 M guanidinium thiocyanate, 45 mM Tris/HCI, 18%, preferably 15% (w/v) Triton-X-100, 0.8% (w/v) DTT and has a pH of 6.0. In a further preferred embodiment, the volume for receiving the blood sample is at a negative pressure which can be adjusted such that a previously determined blood volume is drawn into the vessel after a blood vessel has been pierced. Evacuated vessels of this type are available on the market. The vessel which contains the blood sample can then be immediately subjected to further analyses or, alternatively, may be stored for a long period of time (up to several days) without any disadvantages for the quality of the sample. In the method of the invention, the freshly taken blood is directly contacted in the blood withdrawing vessel with the above-described solution so that all processes which might change the nucleic acid pattern of the sample are immediately halted. Therefore, the data determined at a later time with respect to the detected nucleic acids very accurately represent the actual state at the time of blood withdrawal, i.e. both with respect to the quantities and the types of nucleic acids. Preferably, the blood amount taken is 0.1 to 4 times the solution fed into the vessel. The solution is preferably 0.5 to 5.0 ml. Thus the final concentration of 7 guanidinium salt after blood addition is 1,0 to 5 M, preferably 1 to 3 M, particularly preferred, are 2-3 M (see Example 7). The vessel proposed by the invention is preferably used for blood withdrawal whenever the blood sample is to be used for analyzing nucleic acids. The use of the above-mentioned solution as a component of the described withdrawal system alone guarantees the immediate lysis of the cells and the simultaneous stabilization of the sample by immediate inactivation of the nucleases. Surprisingly, the blood sample obtained thereby can be stored even at room temperature or higher for several days. Moreover, the withdrawal system guarantees contamination-free and non-infectious handling, from sampling through nucleic acid isolation to analysis. In the conventional methods of nucleic acid isolation, additional handling steps have so far been required (e.g. the transfer of the blood sample taken into the reagents for nucleic acid isolation, etc.), which entails an additional risk of infection. The sample obtained with the blood withdrawing system is compatible with all of the conventional standard methods of nucleic acid isolation. In this respect, particular mention should be made of methods based on binding nucleic acids to glass surfaces, although sequence-specific binding to complementary nucleic acid and solvent-based extraction methods may also be used. Thus the invention as described consists of a blood withdrawing system which is conceived such that the following conditions are satisfied. 1. Controlled sampling and simultaneous stabilization of the nucleic acids (DNA. RNA) contained in the sample material. 2. Sampling in which the use of anticoagulants can be completely omitted. 3. The sample obtained by way of the above-described blood withdrawing system can be used universally with all of the known systems for isolating nucleic acids. 4. The blood withdrawing system is stable in storage. 8 Additionally, it has surprisingly been found that the sample obtained by way of the described withdrawal system can be stored in the vessel for a long period of time without degradation of the nucleic acids (see Examples 2,3, 7,8). The following examples will explain the invention: Example 1: The blood withdrawing system may comprise, in a preferred embodiment, the following (see Fig. 1): A small tube is filled with a defined volume of the nucleic acid-stabilizing substance and is placed under a defined vacuum and sealed with a septum. The septum is constructed such that it is compatible with the standard sampling accessories (cannula, etc.). In the present example 2.2 ml reagent was supplied and the vacuum was adjusted such that exactly 2.2 ml blood could flow in during sampling. The nucleic acids contained in the inflowing blood flow were immediately converted into a stable form. General preliminary remark regarding the following examples. In all of the examples described hereinbelow, the nucleic acid-stabilizing substance (N-sS) had, unless indicated otherwise, the following composition: 45 mM Tris, 5 M guanidinium thiocyanate (GTC), 0.8% (w/v) dithiothreitol (DTT), 18% (w/v) Triton-X-100, pH 6.0. In all of the examples described, the nucleic acid-stabilizing substance was, unless indicated otherwise, mixed with the sample in the ratio of 1 to 1 (1 volume N-sS plus 1 volume sample material). A lower concentration of N-sS, e.g. 1 volume N-sS plus 5 volumes sample, might effect a degradation of RNA. 9 Blood was stabilized for all examples by directly introducing the blood upon withdrawal into the small tube mixed with N-sS. Example 2: Stability of nucleic acid after mixing sample material and N-sS. Isolation of RNA and DNA from the sample lysate with silica-derivatized surfaces. Material and method: The sample material for DNA and RNA isolation was used directly after withdrawal, after storage at 4°C for 6 days, and after storage at -20°C for 1 month. The HighPure RNA Isolation Kit (Boehringer Mannheim, cat. no. 1828 665) was used for isolating RNA (Fig. 2). The instructions given in the package leaflet were modified as follows: A volume of 2.4 ml sample lysate was applied in 4 aliquots at 600 ul each to the column, so that a sample material of 2.4 ml lysate was applied on the whole. All of the other steps were carried out in accordance with the package leaflet. The RNA was finally eluted with 100 ul elution buffer. For the isolation of DNA (Fig. 3), the QiaAmp Blood Kit (Qiagen cat. no. 29104) was used. The standard procedure described in the package leaflet was modified in various points: a volume of 400 ul of sample was directly applied to the column; the binding reagent contained in the kit was not used. 25 ul proteinase-K batch solution were added and the sample was incubated at room temperature for 10 min. Subsequently, the column was put into a collection vessel and centrifuged as described in the package leaflet. All of the further steps were carried out in accordance with the description in the package leaflet, except for the use of ethanol. The elution volume was 200 ul. 10 Example 3: Isolation of mRNA from sample lysate using streptavidin-coated magnetic particles and biotin-labeled Oligo(dT) (Fig. 4): Material and method: 20 ml sample lysate were introduced into a vessel. The mRNA was isolated in accordance with the following method: Firstly, 30 ml hybridization buffer (20 mM Tris-HCI, 300 mM NaCI, 6 nM biotin-labeled Oligo(dT), pH 7.4) were added to the lysate. 3 mg streptavidin magnetic particles (Boehringer Mannheim) were then added. The sample was mixed and incubated at room temperature for 5 min. The magnetic particles were separated with the aid of a magnet; the supernatant was discarded. The particles were then resuspended in wash buffer 1(10 mM Tris-HCI, 200 mM NaCI, 1% Triton-X-100, pH 7.5) and washed three times with wash buffer 2 (10 mM Tris-HCI, 200 mM NaCI, pH 7.5) (washing steps: resuspension, magnetic separation, removal of the supernatant). After the last washing step, the supernatant was completely removed and the particles were resuspended in 20 ul distilled water. The sample was heated to 70°C for 5 min. The magnetic particles were separated and the supernatant containing the mRNA was analyzed by means of gel electrophoresis. Example 4: Isolation of DNA and RNA using a modified rule according to Chomczynski and Sacchi (Analytical Biochemistry 162, 156-159 (1987)) (example of a method based on solvent extraction) (Fig. 5): 11 Material and method: 2 ml sample volume were transferred from the blood withdrawing vessel into a small tube. 0.2 ml of a 2 M sodium acetate solution, pH 4, 2 ml phenol (water saturated) and 0.4 ml of a chloroform-isoamyl alcohol mixture (49:1) were then added, the sample being thoroughly mixed after addition of each solution. The complete solution was vigorously shaken for 10 seconds and incubated on ice for 15 minutes. The sample was centrifuged for 20 minutes at 4°C at 10000 g. After centrifugation, the RNA was in the aqueous phase; the DNA and proteins in the intermediate and phenol phase. The aqueous phase was transferred into a new vessel and mixed with 1 ml isopropanol. To precipitate the RNA, the sample was stored at -20°C for 1 houY. After renewed centrifugation at 4°C at 10000 g the RNA was pelleted. The pellet was resuspended in 0.3 ml buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M2-mercaptoisopropanol), transferred into a new 1.5 ml Eppendorf vessel and mixed with 1 volume of isopropanol. After incubation at -20°C for 1 hour, the solution was centrifuged in an Eppendorf centrifuge for 10 minutes at 4°C. The RNA pellet was placed in 75% ethanol and concentrated by centrifugation (Speed vac) and dried. For further processing, the sample was dissolved in 100 ul 10 mM Tris-HCI, pH 6.5. Example 5 Importance of reducing reagents (such as DTT) in the stabilizing solution for the long-term stability of RNA Material and method: Stabilizing solution used: 4.0 M GTC; 13.5% Triton X100; 45 mM Tris/HCI; with or without 120 mM DTT. 700 ul serum were mixed with 700 ul stabilizing solution. 12 After incubation for 2 min 20 ul MS2-RNA (0.8 ug/ul of Roche) were added. The samples were incubated at 40°C for 180 min and then processed in aliquots of 400 ul each using the High Pure total RNA Kit of Roche. The samples were applied in one step to the column without addition of the binding reagent of the kit and centrifuged in accordance with the instructions. The subsequent washing steps and elution of the RNA in 50 ul elution buffer were carried out in accordance with the instructions. The analysis was carried put by means of agarose gel (see Fig. 6). Result: Without the addition of reducing reagents to the stabilizing solution, no long-term stabilization of RNA can be achieved. Example 6 Stability of free MS2-RNA in serum. Kinetics of the RNA degradation by sample components. Material and method: 250 l serum were spiked with 10 ul MS2-RNA (0.8 ug/ul of Roche) and incubated at room temperature. Immediately after the addition of RNA, after 2 min to 50 min, the natural RNA degradation in serum was halted by adding 250 ul stabilizing solution. All batches were analyzed twice. As a standard, one sample was not mixed with MS2-RNA until after the stabilizing solution had been added to the serum and was processed in parallel. All samples were processed in parallel with the High Pure viral RNA Kit of Roche. The samples were applied to the column in one step without adding the binding reagent of the kit and centrifuged according to instructions. The subsequent washing steps and elution of the RNA in 50 ul elution buffer were carried out according to instructions. 20 pi of the eluate were separated by means of a 1.2% native agarose gel and analyzed (see Fig. 7). 13 Result: MS2-RNA is not stable in serum. Already 2 minutes after addition of RNA to the serum, the RNA is completely degraded. By adding stabilizing solution to the serum in a ratio of 1:1, this process can be stopped immediately, and stabilization of the RNA can be achieved at same the time as the stabilizing solution is added (= blood withdrawal). Example 7 Stability of MS2-RNA in serum/stabilizing solution. Dependence on the GTC concentration Material and method Stabilizing solutions used: 3-5 M GTC; 13.5% Triton X100; 50 mM DTT; 42 mM Tris/HCI pH of the solutions: about 4.0 pH of the solutions after addition of serum: about 6.7. 2 ml serum were mixed with 2.5 ml of the respective stabilizing solutions. After an incubation time of 2 to 5 min, 90 ul MS2-RNA (0.8 ug/ul of Roche) were added and incubated at 40°C. 400 ul samples were taken at regular intervals and processed using the High Pure total RNA Kit of Roche according to Example 5. The samples were eluted in 50 ul and frozen at -20°C. For analysis of RNA integrity, 20 ul of the eluate were applied to a 1.5% agarose gel (see Fig. 8). For the PCR analysis of the samples 10 ul of the eluate were reversely transcribed by means of AMV-RT (Roche) and subsequently analyzed by means of PCR on the Lightcycler: AMV-RT buffer dNTP's (final concentration 10 mM) RNase inhibitor (Roche, 20 units) Primer 2827 (final concentration 1 uM) 14 The PCR was carried out on the Lightcycler at an annealing temperature of 61 °C using SYBR-Green as the detection system. Batch for PCR: The amplificate of the PCR was completely applied to a 2% agarose gel (see Fig. 9). Result: RNA integrity at 40°C after 3 days. The agarose gel in Fig. 8 shows 20 ul of the eluted MS2-RNA after incubation at 40°C for 3 days. After this period, distinct differences in the RNA integrity are discernible depending on the GTC content. Thus, a salt content of less than 2 M in the serum/stabilization solution is of advantage in preserving the integrity of the RNA. Amplificability of the RNA at 40°C after 8 days. Although the start of degradation of the RNA was already detected at 40°C after 3 15 days, all of the RNA samples could be amplified after incubation of 8 days at 40°C and clearly detected. The amplificate of the PCR was fully applied to a 2% agarose gel (see Fig. 9). Example 8 Stability of MS2-RNA in serum/stabilizing solution: dependence on the pH of the sample mixed with stabilization solution. Material and method: pH after addition of serum between 6.7 and 8.0 2.5 ml stabilizing solution were mixed with 2.0 ml serum. After adding 90 ul MS2-RNA (0.8 ug/ul, Roche), the samples were incubated at room temperature. The RNA was processed at regular intervals from a 500 ul sample with the Roche viral RNA Kit in accordance with Example 6 and isolated in 50 ul elution buffer. 20 ul of the eluate were analyzed by means of agarose gel (see Fig. 10). Result: The pH of the serum/stabilizing solution and thus the pH and the buffer range of the stabilizing solution are decisive for the long-term stabilization of RNA. Whilst at a pH of 8.0 an intact RNA could no longer be detected already after 2 days, intact RNA is still detectable within a pH range between of 6.6 and 7.0 after 13 days of incubation at room temperature. 16 Apart from the pH, however, an optimally adjusted GTC concentration is also of importance to the long-term stabilization of RNA (see also Example 7). The illustrated example demonstrates that a GTC final concentration in the stabilized sample of 2.2 M GTC is better than 2.78 for long-term stabilization of RNA. Legends Fig. 1: Sampling vessel with N-sS, defined vacuum, sealed with septum. Fig. 2: Gel analysis (1% agarose) of RNA which was stored in the sampling vessel for different periods of time. Column 1: Isolation directly after sampling (no storage), column 2: storage for one month at -20°C, column 3: storage for 6 days at 4°C. The amount of the applied RNA corresponded to a blood volume of 120 ul. Fig. 3: Gel analysis (1% agarose) of DNA which was stored in the sampling vessel for different periods of time. Column 1: isolation directly after sampling (no storage), column 2: storage for one month at -20°C, column 3: storage for 6 days at 4°C. The amount of the applied DNA corresponded to a blood volume of 10 ul. Fig. 4: Gel analysis (1% agarose) of mRNA which was isolated from 10 ml blood (column 2). Molecular weight marker (column 1). In addition to the mRNA, the rRNA bands are visible. The sharp contours of the bands demonstrate the integrity of the nucleic acids. Fig. 5: Gel analysis (1% agarose) of the RNA which was isolated from 120 ul blood. 17 Fig. 6: Gel analysis of isolated MS2-RNA after incubation in serum/stabilizing solution with/without DTT for 180 min at 40°C. Column 1: positive control: MS2-RNA, column 2: DNA marker, column 3,4,5: MS2-RNA after incubation with DTT-eontaining stabilizing solution, column 6,7,8: MS2-RNA after incubation with stabilizing solution without DTT. Fig. 7: Gel analysis of isolated MS2-RNA after incubation in serum for 0-50 min Column 10,17: MS2-RNA standard, column 9,16: DNA marker, column 7,8: incubation for 0 min, column 5,6: incubation for 2 min, column 3,4: incubation for 5 min, column 1,2: incubation for 10 min, column 11,12: incubation for 15 min, column 13,14: incubation for 30 min, column 15: incubation for 50 min Fig. 8: Gel analysis of MS2-RNA which was isolated after incubation in serum/stabilizing solution for 3 days at 40°C. The GTC content of the stabilizing solution after addition of serum in which the relevant RNA sample was incubated is indicated in the corresponding column. Column 1: 2.70 M GTC, column 2: 2.5 M GTC, column 3: 2.36 M GTC, column 4: 2.20 M GTC, column 5: 2.08 M GTC, column 6:1.94 M GTC, column 7: 1.80 M GTC, column 8:1.66 M GTC. 18 Fig. 9: Gel analysis of the PCR amplificates of MS2-RNA which was isolated after 1 day and 8 days, respectively, with incubation at 40°C in serum/stabilizing solution. Column 1: Amplificate of the RNA isolated after 1 day, column 2: amplificate of the RNA isolated after 8 days, column 3: DNA marker, column 4: MS2-RNA positive control: 0.8 ug in 10 ul RT, 1:50 diluted, 1 ul amplified. Fig. 10: Gel analysis of isolated MS2-RNA after 6 days (column 2-12) and 13 days (column 14-19), respectively, with incubation at room temperature in serum/stabilizing solution. The pH obtained after mixing the serum and stabilizing solution is noted after the corresponding columns. Column 1,13, 20: DNA marker, column 2: pH 8.0, column 3: pH 7.7, column 4: pH 7.5, column 5: pH 7.35, column 6: pH 7.18, column 7,14: pH 7.07, column 8,15: pH 6.94, column 9,16: pH 6.8, column 10,17: pH 6.72, column 11,18: pH 6.68 and column 12,19: pH 6.7. The stabilizing solution of RNA in column 12,19 had the same pH as that of the RNA in column 11, but contained 5 M GTC instead of 4 M. We claim ;- 1. A blood withdrawing vessel containing a nucleic acid- stabilizing aqueous solution for stabilizing nucleic acids in"the withdrawn blood directly upon contact with the solution, the solution comprising the following components : a guanidinium salt in a concentration of 1 to 8.0 M; a buffer substance in a concentration of 10 to 300 mM; a reducing agent in a concentration of 0.1 to 10%, by wt; and a detergent in a concentration of 5 to 30%, by wt. 2. The vessel as claimed in claim 1, wherein the guanidinium salt is selected from guanidinium thiocyanate and guanidinium chloride. 3. The vessel as claimed in claim 1, wherein the guanidinium salt is present in a concentration of 2.5 to 8.0 M. 4. The vessel as claimed in claim 1, wherein the buffer substance is selected from Tris, HEPES, MOPS, citrate and phosphate buffer. .5. The vessel as claimed- in claim 1, wherein the detergent is selected from Triton-X-100, NP-40, polydocanol and Tween 20. 6. The vessel as claimed in claim 1, wherein the reducing agent is selected from dithiothreitol, y$-mercapto£thanol and TCEP. 7. "The vessel as claimed in claim 1, wherein the pH of the solution is between 4.0 and 7.5. 19 8. The vessel as claimed, in claim 1, wherein the solution contains the following components : 4m guanidinium thiocyanate; 45 mM Tris/HCL; 15% (w/v) Triton-X-100; 0.8% (w/v) DTT, wherein the pH is at 6.0. 9. The vessel as claimed in claim 1, wherein it has a vacuum in the chamber which is provided for receiving blood. 10. The vessel as claimed in claim 1, wherein it contains withdrawn blood, 11. A method of withdrawing blood in the vessel as claimed in claim l comprising the steps of directly introducing the blood into the said vessel, 12. The method as claimed in claim 11, wherein an amount of blood that is withdrawn is 0.1 to 4 times the volume of the solution in the vessel. 13. The method as claimed in claim 12, wherein the cencentration of the guanidinium salt after the blood is introduced is between 1.0 M and 5 M. 14. The method as claimed in claim 11 wherein the method for stabilizing and/or isolating nucleic acids from blood, comprising the step of introducing blood into the said vessel and, optionally, isolating the nucleic acids with conventional methods. 15. The method as claimed in claim 11, wherein the pH of the solution is adjusted such that, following the introduction of the blood, a pH between 4.0 and 7.5 is obtained. 20 The present invention relates to a vessel for withdrawing blood, the vessel containing a solution which comprises a guanidinium salt, a buffer substance, a reducing agent, and/or a detergent as components. The vessel is particularly well suited to sampling blood which is to be analyzed with respect to nucleic acids.

Full Text

VESSEL FOR WITHDRAWING BLOOD
The present invention relates to a vessel for withdrawing blood, the blood withdrawn being specifically intended for use in stabilizing and analyzing nucleic acids.
When blood is taken, it is normally collected in vessels which already contain anticoagulants such as heoarin. citrate or EDTA. The blood is thereby prevented from coagulating. The blood samples obtained thereby can be stored at suitable temperatures for a long time. This way of obtaining blood has, however, considerable drawbacks if nucleic acids such as (m)RNA or DNA are to be analyzed. For such purposes the nucleic acids contained in the sample should optimally be stabilized immediately at the time of withdrawal, i.e. a degradation of the nucleic acids present should be prevented, but also the new synthesis of mRNA.
This objective of a stable storage of the nucleic acids contained in the sample material, i.e. from the moment of withdrawal, has not been achieved yet in practice for the following reasons:
Cells contain nucleases, enzymes, which destroy nucleic acids as soon as they come into contact with the substrates thereof (RNA, DNA). The effect of cellular and extracellular nucleases is normally under physiological control as long as the cells are in their normal environment. The withdrawal of blood causes more or less significant changes in the nucteiGacMsmDMngd inihe^alls. Nucleases are then released within the cells and/or by the lysis of cells to the outside. Moreover, nucleic acids are synthetized to a greater or lesser degree. In particular the lona-term storage of blood leads to aaina and destruction of the cells
!A

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Another problem arising with long-term storage of blood samples obtained using standard withdrawal methods is the considerable change in the sample material. Such changes, e.g. strong lysis of cells, may have the effect of preventing the standard methods for isolating nucleic acids from functioning in a sufficiently efficient manner.
Apart from the problems relating to stable storage of nucleic acids contained in the sample material, further difficulties arise in the conventional method for withdrawing blood. Conventional anticoagulants are often not separated efficiently enough when isolating nucleic acids and interfere with the subsequent analysis of nucleic acids, e.g. during amplification by means of PCR (polymerase chain reaction). Heparin is e.g. a generally known inhibitor of PCR.
Finally, another issue in the quantitative analysis of nucleic acids is how the method as a whole from sampling through to measuring the nucleic acids can be controlled under standardized conditions. Ideally, a quantitatively and qualitatively defined standard nucleic acid should be added to the sample material during the withdrawal stage and should be subjected to the whole process of sampling and determination. This can also not be accomplished with conventional withdrawal systems.
A further drawback of conventional blood withdrawal is the risk of transferring infectious material because manual process steps have so far been needed in order to isolate the nucleic acids. Contact with potentially infectious germs cannot be ruled out.
The background literature describes a method in which the blood sample is mixed with guanidinium salt directly after withdrawal from a patient (EP 0 818 542 A1). In this method the guanidinium salt is present in powder form in order to take advantage of the greater stability of the guanidinium salt. This method, however,

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has serious drawbacks because, for instance, the salt must first dissolve in the added blood. This dissolution process depends, in particular, on the temperature and cannot be controlled because of the nontransparent sample material used. The use of this type of product for diagnostic medical purposes is thus very problematic.
Furthermore, nucleases are extremely active enzymes which can only be inhibited under extremely denaturing conditions. Denaturation depends on the concentration of the guanidinium salt in solution. The cited method does not specify in any way that the concentration of guanidinium salt in solution should be of a concentration that will have an inhibiting effect. Thus, there is an uncontrolled degradation of nucleic acids during the dissolution process. Moreover, in this method the addition of reducing agents is omitted, without which effective inhibition, in particular of RNases, can not be guaranteed (see Example no. 5).
Moreover, the sample prepared in this way can not directly be used for subsequent isolation of the nucleic acid on glass surfaces. Furthermore, the use of guanidinium salt powder does not permit the addition of internal nucleic acid standards. Such standards are mandatory for process control and exact quantification.
The present invention was based on the technical problem of providing a vessel for withdrawing blood which does not have the drawbacks of the prior art. In particular, it should be possible to subject the sample taken with the vessel directly to the standard methods for analyzing nucleic acids without the need for further sample preparation steps.
In accordance with the invention, this problem is solved by a vessel for withdrawing blood, the vessel containing an aqueous solution comprising the following components:

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a guanidinium salt; a buffer substance; a reducing agent; and/or a detergent.
The vessel of the invention has the following advantages: 1. Blood is already lysed at the moment of withdrawal in that the withdrawal vessel already contains a nucleic acid-stabilizing substance in solution. 2. The nucleic acid-stabilizing substance is composed such that the sample material, in particular the nucleic acids contained therein, are immediately stabilized upon contact with the solution. 3. In contrast to all of the former standard withdrawal systems, such as EDTA or heparin-containing withdrawal vessels, the stabilized sample need no longer be handled as infectious material. 4. The nucleic acid-stabilizing substance is composed such that the sample material can be used directly in subsequent isolating methods. 5. The nucleic acid-stabilizing substance can be separated during subsequent isolation so efficiently that no inhibition of PCR is observed. 6. An internal standard may be added to the nucleic acid-stabilizing substance. This allows the whole method to be controlled, from the moment of sampling up to the detection of nucleic acids.
The withdrawal vessel mentioned under item 1 is a conventional blood withdrawing vessel (small tube) into which a defined volume of a nucleic acid-stabilizing substance is introduced. A small tube is then preferably placed under a defined vacuum which guarantees that only a specific volume of blood can flow thereinto during withdrawal. The small tube can be handled by conventional blood-taking methods. The solution contained in the tube contains the following reagents in a specially preferred embodiment: Guanidinium thiocyanate, Triton-X-100, dithiothreitol and a suitable buffer system, such as citrate, Tris or HEPES. In the described composition the solution is compatible with the vacuum tube. This solution can be stored in the vacuum tube without any problems and without any

5
impairment to the desired stabilizing function. The whole system presents no problems, in particular to blood donors, and is safe during sampling.
The solution containing the guanidinium salt, the buffer substance, the reducing agent and/or the detergent is stable in storage and converts the supplied and freshly taken blood into a material which is also stable in storage and can be transferred directly to standard nucleic-acid analysis kits (e.g. those of Roche or Qiagen).
Guanidinium thiocyanate and/or guanidinium chloride are preferred as the guanidinium salt.
Preferably, the guanidinium salt is present in a concentration of 2.0 to 8.0 M. Tris or citrate is preferred as the buffer substance, the exact pH being preferably adjusted with HCI. Other possible buffers are however HEPES, MOPS, citrate and phosphate buffer, such as PBS.
The buffer concentration is preferably between 10 and 300 mM, particularly preferably between 10 and 100 mM.
Triton-X-100 is preferred as the detergent. Other possible detergents are NP-40, Tween 20, polydocanol or other detergents.
The detergent concentration is preferably 5 to 30% (w/v), particularly preferably 10 to 20% (w/v).
DTT is preferred as the reducing agent, but S-mercaptoethanol, TCEP (Tris(2-carboxyethyl)phosphine) or other reducing agents can also be used.

6
The preferred concentration of the reducing agent is 0.1 to 10% (w/v), particularly preferred are 0.5 to 2% (w/v).
The pH of the solution is preferably 3.0 to 9.0, particularly preferably 4.0 to 7.5,
particularly preferably 5 to 6.
The pH of the solution is chosen in particular such that a pH ranging from 5.0 to
7.6 is set after addition of the sample material. Particularly preferred is a pH
between 6.3 and 6.9 (see Example no. 8).
A particularly preferred solution preferably contains 4 M guanidinium thiocyanate,
45 mM Tris/HCI, 18%, preferably 15% (w/v) Triton-X-100, 0.8% (w/v) DTT and has
a pH of 6.0.
In a further preferred embodiment, the volume for receiving the blood sample is at a negative pressure which can be adjusted such that a previously determined blood volume is drawn into the vessel after a blood vessel has been pierced. Evacuated vessels of this type are available on the market.
The vessel which contains the blood sample can then be immediately subjected to further analyses or, alternatively, may be stored for a long period of time (up to several days) without any disadvantages for the quality of the sample.
In the method of the invention, the freshly taken blood is directly contacted in the blood withdrawing vessel with the above-described solution so that all processes which might change the nucleic acid pattern of the sample are immediately halted. Therefore, the data determined at a later time with respect to the detected nucleic acids very accurately represent the actual state at the time of blood withdrawal, i.e. both with respect to the quantities and the types of nucleic acids.
Preferably, the blood amount taken is 0.1 to 4 times the solution fed into the vessel. The solution is preferably 0.5 to 5.0 ml. Thus the final concentration of

7
guanidinium salt after blood addition is 1,0 to 5 M, preferably 1 to 3 M, particularly preferred, are 2-3 M (see Example 7).
The vessel proposed by the invention is preferably used for blood withdrawal whenever the blood sample is to be used for analyzing nucleic acids.
The use of the above-mentioned solution as a component of the described withdrawal system alone guarantees the immediate lysis of the cells and the simultaneous stabilization of the sample by immediate inactivation of the nucleases. Surprisingly, the blood sample obtained thereby can be stored even at room temperature or higher for several days. Moreover, the withdrawal system guarantees contamination-free and non-infectious handling, from sampling through nucleic acid isolation to analysis. In the conventional methods of nucleic acid isolation, additional handling steps have so far been required (e.g. the transfer of the blood sample taken into the reagents for nucleic acid isolation, etc.), which entails an additional risk of infection.
The sample obtained with the blood withdrawing system is compatible with all of the conventional standard methods of nucleic acid isolation. In this respect, particular mention should be made of methods based on binding nucleic acids to glass surfaces, although sequence-specific binding to complementary nucleic acid and solvent-based extraction methods may also be used.
Thus the invention as described consists of a blood withdrawing system which is conceived such that the following conditions are satisfied. 1. Controlled sampling and simultaneous stabilization of the nucleic acids (DNA. RNA) contained in the sample material. 2. Sampling in which the use of anticoagulants can be completely omitted. 3. The sample obtained by way of the above-described blood withdrawing system can be used universally with all of the known systems for isolating nucleic acids. 4. The blood withdrawing system is stable in storage.

8
Additionally, it has surprisingly been found that the sample obtained by way of the described withdrawal system can be stored in the vessel for a long period of time without degradation of the nucleic acids (see Examples 2,3, 7,8).
The following examples will explain the invention: Example 1:
The blood withdrawing system may comprise, in a preferred embodiment, the following (see Fig. 1): A small tube is filled with a defined volume of the nucleic acid-stabilizing substance and is placed under a defined vacuum and sealed with a septum. The septum is constructed such that it is compatible with the standard sampling accessories (cannula, etc.). In the present example 2.2 ml reagent was supplied and the vacuum was adjusted such that exactly 2.2 ml blood could flow in during sampling. The nucleic acids contained in the inflowing blood flow were immediately converted into a stable form.
General preliminary remark regarding the following examples.
In all of the examples described hereinbelow, the nucleic acid-stabilizing substance (N-sS) had, unless indicated otherwise, the following composition: 45 mM Tris, 5 M guanidinium thiocyanate (GTC), 0.8% (w/v) dithiothreitol (DTT), 18% (w/v) Triton-X-100, pH 6.0.
In all of the examples described, the nucleic acid-stabilizing substance was, unless indicated otherwise, mixed with the sample in the ratio of 1 to 1 (1 volume N-sS plus 1 volume sample material). A lower concentration of N-sS, e.g. 1 volume N-sS plus 5 volumes sample, might effect a degradation of RNA.

9
Blood was stabilized for all examples by directly introducing the blood upon withdrawal into the small tube mixed with N-sS.
Example 2:
Stability of nucleic acid after mixing sample material and N-sS. Isolation of RNA and DNA from the sample lysate with silica-derivatized surfaces.
Material and method:
The sample material for DNA and RNA isolation was used directly after withdrawal, after storage at 4°C for 6 days, and after storage at -20°C for 1 month.
The HighPure RNA Isolation Kit (Boehringer Mannheim, cat. no. 1828 665) was used for isolating RNA (Fig. 2). The instructions given in the package leaflet were modified as follows: A volume of 2.4 ml sample lysate was applied in 4 aliquots at 600 ul each to the column, so that a sample material of 2.4 ml lysate was applied on the whole. All of the other steps were carried out in accordance with the package leaflet. The RNA was finally eluted with 100 ul elution buffer.
For the isolation of DNA (Fig. 3), the QiaAmp Blood Kit (Qiagen cat. no. 29104) was used. The standard procedure described in the package leaflet was modified in various points: a volume of 400 ul of sample was directly applied to the column; the binding reagent contained in the kit was not used. 25 ul proteinase-K batch solution were added and the sample was incubated at room temperature for 10 min. Subsequently, the column was put into a collection vessel and centrifuged as described in the package leaflet. All of the further steps were carried out in accordance with the description in the package leaflet, except for the use of ethanol. The elution volume was 200 ul.

10
Example 3:
Isolation of mRNA from sample lysate using streptavidin-coated magnetic particles and biotin-labeled Oligo(dT) (Fig. 4):
Material and method:
20 ml sample lysate were introduced into a vessel. The mRNA was isolated in accordance with the following method: Firstly, 30 ml hybridization buffer (20 mM Tris-HCI, 300 mM NaCI, 6 nM biotin-labeled Oligo(dT), pH 7.4) were added to the lysate.
3 mg streptavidin magnetic particles (Boehringer Mannheim) were then added. The sample was mixed and incubated at room temperature for 5 min. The magnetic particles were separated with the aid of a magnet; the supernatant was discarded. The particles were then resuspended in wash buffer 1(10 mM Tris-HCI, 200 mM NaCI, 1% Triton-X-100, pH 7.5) and washed three times with wash buffer 2 (10 mM Tris-HCI, 200 mM NaCI, pH 7.5) (washing steps: resuspension, magnetic separation, removal of the supernatant). After the last washing step, the supernatant was completely removed and the particles were resuspended in 20 ul distilled water. The sample was heated to 70°C for 5 min. The magnetic particles were separated and the supernatant containing the mRNA was analyzed by means of gel electrophoresis.
Example 4:
Isolation of DNA and RNA using a modified rule according to Chomczynski and Sacchi (Analytical Biochemistry 162, 156-159 (1987)) (example of a method based on solvent extraction) (Fig. 5):

11
Material and method:
2 ml sample volume were transferred from the blood withdrawing vessel into a small tube. 0.2 ml of a 2 M sodium acetate solution, pH 4, 2 ml phenol (water saturated) and 0.4 ml of a chloroform-isoamyl alcohol mixture (49:1) were then added, the sample being thoroughly mixed after addition of each solution. The complete solution was vigorously shaken for 10 seconds and incubated on ice for 15 minutes. The sample was centrifuged for 20 minutes at 4°C at 10000 g. After centrifugation, the RNA was in the aqueous phase; the DNA and proteins in the intermediate and phenol phase. The aqueous phase was transferred into a new vessel and mixed with 1 ml isopropanol. To precipitate the RNA, the sample was stored at -20°C for 1 houY. After renewed centrifugation at 4°C at 10000 g the RNA was pelleted. The pellet was resuspended in 0.3 ml buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M2-mercaptoisopropanol), transferred into a new 1.5 ml Eppendorf vessel and mixed with 1 volume of isopropanol. After incubation at -20°C for 1 hour, the solution was centrifuged in an Eppendorf centrifuge for 10 minutes at 4°C. The RNA pellet was placed in 75% ethanol and concentrated by centrifugation (Speed vac) and dried. For further processing, the sample was dissolved in 100 ul 10 mM Tris-HCI, pH 6.5.
Example 5
Importance of reducing reagents (such as DTT) in the stabilizing solution for the long-term stability of RNA
Material and method:
Stabilizing solution used: 4.0 M GTC; 13.5% Triton X100; 45 mM Tris/HCI; with or without 120 mM DTT. 700 ul serum were mixed with 700 ul stabilizing solution.

12
After incubation for 2 min 20 ul MS2-RNA (0.8 ug/ul of Roche) were added. The
samples were incubated at 40°C for 180 min and then processed in aliquots of
400 ul each using the High Pure total RNA Kit of Roche. The samples were
applied in one step to the column without addition of the binding reagent of the kit
and centrifuged in accordance with the instructions. The subsequent washing
steps and elution of the RNA in 50 ul elution buffer were carried out in accordance
with the instructions.
The analysis was carried put by means of agarose gel (see Fig. 6).
Result: Without the addition of reducing reagents to the stabilizing solution, no
long-term stabilization of RNA can be achieved.
Example 6
Stability of free MS2-RNA in serum. Kinetics of the RNA degradation by sample components.
Material and method:
250 l serum were spiked with 10 ul MS2-RNA (0.8 ug/ul of Roche) and incubated at room temperature. Immediately after the addition of RNA, after 2 min to 50 min, the natural RNA degradation in serum was halted by adding 250 ul stabilizing solution. All batches were analyzed twice. As a standard, one sample was not mixed with MS2-RNA until after the stabilizing solution had been added to the serum and was processed in parallel.
All samples were processed in parallel with the High Pure viral RNA Kit of Roche. The samples were applied to the column in one step without adding the binding reagent of the kit and centrifuged according to instructions. The subsequent washing steps and elution of the RNA in 50 ul elution buffer were carried out according to instructions.
20 pi of the eluate were separated by means of a 1.2% native agarose gel and analyzed (see Fig. 7).

13
Result: MS2-RNA is not stable in serum. Already 2 minutes after addition of RNA to the serum, the RNA is completely degraded. By adding stabilizing solution to the serum in a ratio of 1:1, this process can be stopped immediately, and stabilization of the RNA can be achieved at same the time as the stabilizing solution is added (= blood withdrawal).
Example 7
Stability of MS2-RNA in serum/stabilizing solution. Dependence on the GTC concentration
Material and method
Stabilizing solutions used: 3-5 M GTC; 13.5% Triton X100; 50 mM DTT; 42 mM
Tris/HCI
pH of the solutions: about 4.0
pH of the solutions after addition of serum: about 6.7.
2 ml serum were mixed with 2.5 ml of the respective stabilizing solutions. After an incubation time of 2 to 5 min, 90 ul MS2-RNA (0.8 ug/ul of Roche) were added and incubated at 40°C. 400 ul samples were taken at regular intervals and processed using the High Pure total RNA Kit of Roche according to Example 5. The samples were eluted in 50 ul and frozen at -20°C. For analysis of RNA integrity, 20 ul of the eluate were applied to a 1.5% agarose gel (see Fig. 8).
For the PCR analysis of the samples 10 ul of the eluate were reversely transcribed by means of AMV-RT (Roche) and subsequently analyzed by means of PCR on the Lightcycler:

AMV-RT buffer
dNTP's (final concentration 10 mM) RNase inhibitor (Roche, 20 units) Primer 2827 (final concentration 1 uM)

14

The PCR was carried out on the Lightcycler at an annealing temperature of 61 °C using SYBR-Green as the detection system. Batch for PCR:

The amplificate of the PCR was completely applied to a 2% agarose gel (see Fig. 9).
Result:
RNA integrity at 40°C after 3 days.
The agarose gel in Fig. 8 shows 20 ul of the eluted MS2-RNA after incubation at
40°C for 3 days. After this period, distinct differences in the RNA integrity are
discernible depending on the GTC content. Thus, a salt content of less than 2 M in
the serum/stabilization solution is of advantage in preserving the integrity of the
RNA.
Amplificability of the RNA at 40°C after 8 days.
Although the start of degradation of the RNA was already detected at 40°C after 3

15
days, all of the RNA samples could be amplified after incubation of 8 days at 40°C
and clearly detected.
The amplificate of the PCR was fully applied to a 2% agarose gel (see Fig. 9).
Example 8
Stability of MS2-RNA in serum/stabilizing solution: dependence on the pH of the sample mixed with stabilization solution.
Material and method:

pH after addition of serum between 6.7 and 8.0
2.5 ml stabilizing solution were mixed with 2.0 ml serum. After adding 90 ul MS2-RNA (0.8 ug/ul, Roche), the samples were incubated at room temperature. The RNA was processed at regular intervals from a 500 ul sample with the Roche viral RNA Kit in accordance with Example 6 and isolated in 50 ul elution buffer. 20 ul of the eluate were analyzed by means of agarose gel (see Fig. 10).
Result:
The pH of the serum/stabilizing solution and thus the pH and the buffer range of the stabilizing solution are decisive for the long-term stabilization of RNA. Whilst at a pH of 8.0 an intact RNA could no longer be detected already after 2 days, intact RNA is still detectable within a pH range between of 6.6 and 7.0 after 13 days of incubation at room temperature.

16
Apart from the pH, however, an optimally adjusted GTC concentration is also of importance to the long-term stabilization of RNA (see also Example 7). The illustrated example demonstrates that a GTC final concentration in the stabilized sample of 2.2 M GTC is better than 2.78 for long-term stabilization of RNA.
Legends
Fig. 1:
Sampling vessel with N-sS, defined vacuum, sealed with septum.
Fig. 2:
Gel analysis (1% agarose) of RNA which was stored in the sampling vessel for different periods of time. Column 1: Isolation directly after sampling (no storage), column 2: storage for one month at -20°C, column 3: storage for 6 days at 4°C. The amount of the applied RNA corresponded to a blood volume of 120 ul.
Fig. 3:
Gel analysis (1% agarose) of DNA which was stored in the sampling vessel for different periods of time. Column 1: isolation directly after sampling (no storage), column 2: storage for one month at -20°C, column 3: storage for 6 days at 4°C. The amount of the applied DNA corresponded to a blood volume of 10 ul.
Fig. 4:
Gel analysis (1% agarose) of mRNA which was isolated from 10 ml blood (column 2). Molecular weight marker (column 1). In addition to the mRNA, the rRNA bands are visible. The sharp contours of the bands demonstrate the integrity of the nucleic acids.
Fig. 5:
Gel analysis (1% agarose) of the RNA which was isolated from 120 ul blood.

17
Fig. 6:
Gel analysis of isolated MS2-RNA after incubation in serum/stabilizing solution with/without DTT for 180 min at 40°C.
Column 1: positive control: MS2-RNA, column 2: DNA marker, column 3,4,5: MS2-RNA after incubation with DTT-eontaining stabilizing solution, column 6,7,8: MS2-RNA after incubation with stabilizing solution without DTT.
Fig. 7:
Gel analysis of isolated MS2-RNA after incubation in serum for 0-50 min
Column 10,17: MS2-RNA standard, column 9,16: DNA marker, column 7,8: incubation for 0 min, column 5,6: incubation for 2 min, column 3,4: incubation for 5 min, column 1,2: incubation for 10 min, column 11,12: incubation for 15 min, column 13,14: incubation for 30 min, column 15: incubation for 50 min
Fig. 8:
Gel analysis of MS2-RNA which was isolated after incubation in serum/stabilizing solution for 3 days at 40°C. The GTC content of the stabilizing solution after addition of serum in which the relevant RNA sample was incubated is indicated in the corresponding column.
Column 1: 2.70 M GTC, column 2: 2.5 M GTC, column 3: 2.36 M GTC, column 4: 2.20 M GTC, column 5: 2.08 M GTC, column 6:1.94 M GTC, column 7: 1.80 M GTC, column 8:1.66 M GTC.

18
Fig. 9:
Gel analysis of the PCR amplificates of MS2-RNA which was isolated after 1 day and 8 days, respectively, with incubation at 40°C in serum/stabilizing solution.
Column 1: Amplificate of the RNA isolated after 1 day, column 2: amplificate of the RNA isolated after 8 days, column 3: DNA marker, column 4: MS2-RNA positive control: 0.8 ug in 10 ul RT, 1:50 diluted, 1 ul amplified.
Fig. 10:
Gel analysis of isolated MS2-RNA after 6 days (column 2-12) and 13 days (column 14-19), respectively, with incubation at room temperature in serum/stabilizing solution. The pH obtained after mixing the serum and stabilizing solution is noted after the corresponding columns.
Column 1,13, 20: DNA marker, column 2: pH 8.0, column 3: pH 7.7, column 4: pH 7.5, column 5: pH 7.35, column 6: pH 7.18, column 7,14: pH 7.07, column 8,15: pH 6.94, column 9,16: pH 6.8, column 10,17: pH 6.72, column 11,18: pH 6.68 and column 12,19: pH 6.7. The stabilizing solution of RNA in column 12,19 had the same pH as that of the RNA in column 11, but contained 5 M GTC instead of 4 M.

We claim ;-
1. A blood withdrawing vessel containing a nucleic acid-
stabilizing aqueous solution for stabilizing nucleic acids in"the
withdrawn blood directly upon contact with the solution, the
solution comprising the following components :
a guanidinium salt in a concentration of 1 to 8.0 M; a buffer substance in a concentration of 10 to 300 mM; a reducing agent in a concentration of 0.1 to 10%, by wt; and
a detergent in a concentration of 5 to 30%, by wt.
2. The vessel as claimed in claim 1, wherein the guanidinium
salt is selected from guanidinium thiocyanate and guanidinium
chloride.
3. The vessel as claimed in claim 1, wherein the guanidinium
salt is present in a concentration of 2.5 to 8.0 M.
4. The vessel as claimed in claim 1, wherein the buffer
substance is selected from Tris, HEPES, MOPS, citrate and
phosphate buffer.
.5. The vessel as claimed- in claim 1, wherein the detergent is selected from Triton-X-100, NP-40, polydocanol and Tween 20.
6. The vessel as claimed in claim 1, wherein the reducing agent
is selected from dithiothreitol, y$-mercapto£thanol and TCEP.
7. "The vessel as claimed in claim 1, wherein the pH of the
solution is between 4.0 and 7.5.
19

8. The vessel as claimed, in claim 1, wherein the solution

contains the following components : 4m guanidinium thiocyanate; 45 mM Tris/HCL; 15% (w/v) Triton-X-100; 0.8% (w/v) DTT, wherein the pH is at 6.0.
9. The vessel as claimed in claim 1, wherein it has a vacuum in
the chamber which is provided for receiving blood.
10. The vessel as claimed in claim 1, wherein it contains
withdrawn blood,
11. A method of withdrawing blood in the vessel as claimed in
claim l comprising the steps of directly introducing the blood
into the said vessel,
12. The method as claimed in claim 11, wherein an amount of
blood that is withdrawn is 0.1 to 4 times the volume of the
solution in the vessel.
13. The method as claimed in claim 12, wherein the cencentration
of the guanidinium salt after the blood is introduced is between
1.0 M and 5 M.

14. The method as claimed in claim 11 wherein the method for
stabilizing and/or isolating nucleic acids from blood, comprising
the step of introducing blood into the said vessel and,
optionally, isolating the nucleic acids with conventional
methods.
15. The method as claimed in claim 11, wherein the pH of the
solution is adjusted such that, following the introduction of the
blood, a pH between 4.0 and 7.5 is obtained.

20

The present invention relates to a vessel for withdrawing blood, the vessel containing a solution which comprises a guanidinium salt, a buffer substance, a reducing agent, and/or a detergent as components. The vessel is particularly well suited to sampling blood which is to be analyzed with respect to nucleic acids.

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Patent Number

207201

Indian Patent Application Number

IN/PCT/2001/00277/KOL

PG Journal Number

22/2007

Publication Date

01-Jun-2007

Grant Date

31-May-2007

Date of Filing

08-Mar-2001

Name of Patentee

PREANALYTIX GMBH.

Applicant Address

8634 HOMBRECHTIKON SWITZERLAND

Inventors:

#	Inventor's Name	Inventor's Address
1	HELFTENBEIN ELKE	LEONORENSTRASSE 7 D-70597 STUTTGART,GERMANY

PCT International Classification Number

C12N 15/10

PCT International Application Number

PCT/EP99/05857

PCT International Filing date

1999-08-12

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	19836559.4	1998-08-12	Germany