Chemistry: analytical and immunological testing – Including sample preparation – Stabilizing or preserving
Reexamination Certificate
2000-11-08
2003-08-05
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Stabilizing or preserving
C436S008000, C436S018000, C435S002000, C435S006120, C435S260000, C435S287200, C422S091000, C422S105000, C252S408100
Reexamination Certificate
active
06602718
ABSTRACT:
FIELD OF THE INVENTION
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
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 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 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 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
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.
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.
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.
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.
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.
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.
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.
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.
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 resultin
Augello Frank A.
Bastian Helge
Oelmüller Uwe
Rainen Lynne
Walenciak Matthew
Becton Dickinson and Company
Roylance Abrams Berdo & Goodman L.L.P.
Wallenhorst Maureen M.
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