Biological sample collection and preservation

Abstract

An embodiment of the claimed invention is directed to a method that greatly streamlines and reduces costs for tissue preparation, preservation, long-term storage and sample retrieval for molecular analysis using a method based on dried blood spot (DBS) technology. In this method, a small needle punch sample of freshly excised tissue will be homogenized in stabilizing reagent and inserted into a device containing absorbent material and drying agent. This device is suitable for long-term sample storage at ambient temperature and allows for easy removal of sections for biomarker analysis.

Claims

1. A method of extracting and storing a biological sample with a device, the method comprising: retrieving into the device a tissue sample from a specimen by extending a first plunger of the device; contacting the tissue sample with a buffer; homogenizing the tissue sample within the device by actuating a second plunger of the device to move a homogenization device attached to the second plunger to produce a liquid homogenate within the device; transferring the liquid homogenate on to an absorbent material; and drying the liquid homogenate on the absorbent material to create a storable sample at ambient temperature.

2. The method of claim 1, further comprising the step of subjecting a portion of the storable sample to testing.

3. The method of claim 1, wherein the homogenization device is selected from the group consisting of a grating device, a friction-based device, and a blade-based device.

4. The method of claim 1, wherein the buffer includes at least one aqueous buffer selected from the group consisting of: a phosphate buffer, a PBS containing protease inhibitor, an RNAse inhibitor, a detergent, and an organic solvent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is directed to a device in accordance with an embodiment of the invention;

(2) FIG. 2 is directed to a device in accordance with an embodiment of the invention;

(3) FIG. 3 shows a process flow of an exemplary method in accordance with an embodiment of the invention;

(4) FIG. 4 shows a drawing of the HemaSpot cartridge;

(5) FIG. 5 shows comparison of enzymatic activity from wet and dried tissue homogenate;

(6) FIG. 6 shows comparison of p53 mRNA by RT-PCR from wet and dried tissue homogenate; and

(7) FIG. 7 shows relative quantitation by RT-PCR for mRNA and RNA from wet and dried samples.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

(8) An embodiment of the invention is directed to the sampling, processing and storage of tissue specimens.

(9) In accordance with an embodiment of the invention, a tube-like device is used to sample a portion of a tissue specimen. In this embodiment, the device is adapted to contain a needle that is used to punch or collect a sample of a tissue specimen. In certain embodiments of the invention, the size of the sample that is collected with the device is around 3 mm However, it should be recognized that the size of the sample will largely be dependent upon the uses that the sampled tissue specimen will be subjected to. In certain embodiments of the invention the tube-like device is a syringe or a syringe-like device.

(10) As shown in FIG. 1, a device 10 in accordance with an embodiment of the invention comprises a tube (2) that is adapted to have a needle (1) at its proximate end and a plunger at its distal end. In certain embodiments of the invention, the plunger that is used is a double plunger, which comprises first (4) and second (5) plungers that slide in opposite directions within the syringe. The device is constructed so that only one plunger can be employed at a time. The first plunger (4) is designed to aid in the collection of the tissue specimen. The device further comprises a grating plate (3) that is located within the chamber of the tube 2 at a location that is distal to the location of the needle. The grating plate is distally connected to the second plunger (5), which aids in the homogenization of the collected sample. In other embodiments of the invention, the homogenization step is carried out by friction-based homogenization or blade-type homogenization (i.e. a blender or chopper). In certain embodiments of the invention, the first plunger 4 is an outer plunger and the second plunger 5 is an inner plunger. In certain embodiments of the invention, the tube 2 can be a cylindrical tube or a syringe.

(11) FIG. 2 shows a cross-section of the device 10 set forth in FIG. 1 with the various components labeled similarly to FIG. 1.

(12) As shown in FIG. 3, in an embodiment of the invention, a sample of a tissue specimen is retrieved by using the needle (1) to punch the specimen and extract a sample by using the first plunger (4) to draw the sample into the tube (2). After the sample has been extracted and drawn into the syringe, a stabilizing buffer is introduced to the sample. The buffer can be introduced to the sample by withdrawing it from a buffer source using the first plunger (4). After the buffer has contacted the sample, the grating plate (3) is used to homogenize the sample using the second plunger (5). Examples of the homogenization buffer include aqueous buffers such as phosphate buffers and PBS containing protease inhibitors, RNAse inhibitors, detergents and organic solvents.

(13) Following the homogenization step, the liquid homogenate is stored in a manner that facilitates future testing of the samples. In an embodiment of the invention, the liquid homogenate is applied to a surface of an absorbent material. The purpose of applying the liquid homogenate to the absorbent material is to enable the absorbent material to function as a storage medium for the homogenized sample.

(14) In an embodiment of the invention, the absorbent material containing the liquid homogenate is transferred to a receptacle containing a drying agent. The drying agent facilitates the removal of moisture from the absorbent material. Upon removal of the moisture from the sample, the specimen sample is adsorbed on the surface of the absorbent material and can be stored at ambient temperature. The dried absorbent material can now be used for testing of the specimen.

(15) In an embodiment of the invention, the liquid homogenate is applied to a collection cartridge (FIG. 4). In an embodiment of the invention, the collection cartridge is the HemaSpot™ collection device. This device comprises absorbent paper that is adjacent to a dessicant that permits the drying the sample on the absorbent paper.

(16) The remaining excised tissue is examined by traditional histological or other methods requiring intact tissue and cellular structure.

(17) Biological samples that can be manipulated with the device of the claimed invention include, but are not limited to: 1. Biopsied tissue including tumors of liver, skin, kidney, heart, brain, ovaries, and prostate. 2. Fecal matter (to identify intestinal flora) 3. Plant tissue (i.e. food: vegetables, fruits; crops, trees, flowers)—to identify species, phenotype, viral/bacterial infection 4. Meat—identify species, contamination 5. Forensic tissue—identify DNA; and 5. Soil samples

(18) Success of this dried tissue spot method would be a significant improvement over current tissue methods. Possible advantages as compared to current FFPE and flash freezing methods are outlined in Table 1 below.

(19) TABLE-US-00001 TABLE 1 Feature FFPE Flash Freeze Dried Tissue 1. Non-hazardous sample prep x x ✓ 2. Minimum infrastructure needs x x ✓ 3. Prep time <60 minutes x ✓ ✓ 4. Storage at ambient temp. ✓ x ✓ 5. Simple sample shipment x x ✓ 6. Efficient protein recovery x ✓ ✓ 7. Efficient nucleotide recovery x ✓ ✓ 8. Ease of sample prep & analysis x x ✓

WORKING EXAMPLES

(20) Rat liver tissue (approx. 50 mg) was homogenized in two separate reagent mixtures, A: phosphate buffer saline with 0.5% Triton and B: 10 mM Tris, 1 mM EDTA with 0.5% Triton-X100. Enzymatic activity was compared for homogenate samples that remained wet and equivalent samples that were dried on filter paper (FIG. 5). Activities of alpha-galactosidase (alpha-GAL) and beta-galactosidase (beta-GAL) were measured by incubating at 37° C. for 2 hours with 4-methylumbelliferyl-alpha-D-galactopyranoside and 4-methylumbelliferyl-beta-D-galactopyranoside, respectively. 4-methylumbelliferone (4-MU) formation was measured by fluorescence. Activity levels were similar between wet and dried homogenization samples.

(21) Three separate reagent mixtures were used to homogenize rat liver tissue, A: nuclease-free water B: nuclease-free water with 0.5% CHAPS and C: nuclease-free water with 0.5% Triton-X100. Portions of the homogenates were frozen while separate portions were applied to HemaSpot Devices and allowed to dry. Total RNA was isolated by standard Trizol methods and p53 mRNA levels for wet and dried homogenate were compared by RT-PCR using GAPDH as a reference gene (FIG. 6). Relative p53 mRNA levels were comparable between wet and dried samples on filter paper.

(22) Relative levels of mRNA Auf1 and Lcn2 and RNA Actin from homogenized rat liver tissue was measured by RT-PCR, using GAPDH as a reference gene (FIG. 7). Total RNA was isolated using standard Trizol methods from wet and filter-paper dried homogenates. Dried samples showed equal or higher relative levels of mRNA and RNA as compared to correlating wet samples.

(23) The data set forth in FIGS. 5, 6 and 7 demonstrate the feasibility for accurately measuring protein and nucleotide levels from homogenates of tissue samples that have been dried on filter paper.

(24) Together, these innovations will provide ideal preparation and storage of tissue specimens with minimal processing and refrigeration, while maintaining sample integrity for biomarker analysis. A commercial kit containing all required components would greatly simplify tissue collection and storage. The invention further provides a simplified, low cost tissue specimen preparation and storage method with minimal processing and refrigeration while maintaining sample integrity for analysis of biomarkers such as mRNA, miRNA, DNA, proteins and small molecules. A readily available commercial kit containing all required components would enable collection and analysis of tissues from remote and low resource areas, democratizing biospecimen collection and analysis. Availability of these stable, dried samples allow for simplified analysis of biomarkers to direct cancer treatments for the highest benefit and long-term storage in biorepositories would greatly aid disease and cancer research.

(25) In the preceding detailed description, the invention is described with reference to specific exemplary embodiments thereof. Various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention as set forth in the claims. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.