The present invention is directed to a method and device for collecting, storing, transporting and stabilizing 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 and for inhibiting ex vivo gene induction and degradation during storage.
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.
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 changes of physical conditions such as, for example, temperature 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.
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.
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, investigate and diagnostic metabolic diseases, investigate influence of drugs on gene expression in patients, investigate side and toxic effects of drugs, and to determine the HLA type or other marker of identity.
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.
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.
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.
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.
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 changes in physical conditions such as temperature 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.
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 and immediately contacting the sample with a stabilizing additive to block ex vivo gene induction in the sample, thereby preserving the in vivo transcription profile.
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 by inhibiting or blocking gene induction in the sample during storage. The stabilizing additive is present in an effective amount to stabilize the nucleic acids, particularly RNA, and inhibit or block gene induction.
One aspect of the invention is to prepare a biological sample that is stable at room temperature for extended periods of time with little or no occurrence of gene induction. Accordingly, a method is provided for producing a biological sample that is stable at room temperature with little or no incidence of gene induction during storage.
A further aspect of the invention is to provide a method and device for inhibiting gene induction of nucleic acids in a biological sample and to lyse cells, bacteria, viruses and reticulocytes.
Another aspect 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 gene induction blocking agent.
A further aspect of the invention is to provide a method for stabilizing a biological sample, and particularly whole blood, immediately upon collection from the patient to inhibit or prevent gene induction when the sample is stored at room temperature.
A further aspect of the invention is to provide a method for stabilizing a biological sample, and particularly whole blood, immediately upon collection from the patient to inhibit or prevent gene induction or degradation of nucleic acids when the stabilized blood sample is stored at temperatures below room temperature, typically 2xc2x0 C. to about 8xc2x0 C., or at temperatures suitable for archiving the samples, for example, at temperatures of xe2x88x9220xc2x0 C. to xe2x88x9280xc2x0 C. Frozen samples can be thawed at room temperature for isolation of nucleic acids.
Still another aspect of the invention is to provide a method for blocking ex vivo gene induction in a biological sample immediately on collection of the biological sample.
Another aspect of the invention is to provide an evacuated container that is pre-filled with an effective amount of a gene induction blocking agent, where the container has an internal pressure sufficiently low to draw a predetermined volume of a biological sample into the container.
A further aspect of the invention is to provide a blood collection container for collecting an amount of blood and mixing the blood with a gene induction blocking agent at the point of collection to produce a biological sample that is stable at room temperature by preventing gene induction such that nucleic acid analysis of the sample can be conducted at a later time.
Another aspect of the invention is to provide a method of stabilizing blood by collecting the blood sample in a container having a gene induction blocking agent and a buffer. The gene induction agent can be a detergent, a chaotropic salt, RNase inhibitors, chelating agents, or mixtures thereof. The pH of the resulting mixture is adjusted to inhibit or block the nucleic acid degradation or gene induction and promote efficient recovery of the analyte.
Still another aspect of the invention is to provide a method of stabilizing a blood sample in a collection device at about pH 2 to about pH 5 in the presence of at least one gene induction blocking agent.
The aspects 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 gene induction blocking agent in an effective amount to preserve the biological sample and block or inhibit ex vivo gene induction. The container can be pre-filled with the stabilizing agent.
The aspects of the invention are further attained by providing a method of preparing a room temperature stable biological sample comprising the steps of: providing a sample collection container having a side wall, and a bottom defining an internal chamber where the container contains at least one gene induction blocking agent in an amount and concentration sufficient to block ex vivo gene induction and preserve a biological sample. A biological sample is obtained and immediately introduced into the container and the biological sample is mixed with the gene induction blocking agent to form a stabilized biological sample.
The aspects 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 is pre-filled with 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.
These aspects, advantages and other salient features of the invention will become apparent from the annexed drawing and the following detailed description of the invention.
USPTO Backgrounds
USPTO Backgrounds