Patent Application
20170233792
Traditional methods of cell fixation for urine samples are problematic for both biological and safety reasons. Prior methods have resulted in genetic and protein cross-linking, thereby rendering the single cells nonviable for downstream analysis. Further, the previous fixation methods are chemically volatile and flammable, presenting a safety hazard.
Urine sample preparation is a key factor in obtaining adequate and representative cells for downstream analysis. Current methodology is usually a centrifugation for separation of tumor cells from urine based on their densities. However, this method often collects all types of cells and particles, and the recovery of the cells for subsequent molecular analysis is limited. Recent advances in size-selective microfiltration technology provide an alternative approach for isolation of tumor cells from urine sample.
Analysis of cells in the urine is often necessary in the diagnosis and treatment of urological cancers. In such analyses, cell morphology, protein expression and molecular alternations of the tumor cells are frequently targeted for detection. However, if urine is permitted to be collected at a clinical site and then transported to a central lab, the urine sample will often be stored at either room temperature or 4° C. for a prolonged period of hours before subsequent analysis can be completed. As a result, cells in urine will lyse or undergo apoptosis. In addition, the chemical composition of the urine sample is frequently altered upon standing as a result of environmental changes, for example, temperature change and protease digestion. Furthermore, bacterial contamination and other microorganisms may grow in urine, which may alter microfiltration and downstream analysis.
There remains a need for additional compositions and methods for preserving cells in the urine to enable proper analysis.
Fixation is needed for preserving the genetic material of cells for downstream analysis. The quality and integrity of cells of clinical samples are crucial for sensitivity and accuracy of clinical single cell analysis. In many occasions, the clinical samples may not be processed for single cell analysis immediately and the single cells would degrade dramatically. Traditional fixation methodologies would either induce cross-link between genetic materials and proteins affecting the downstream analysis or be chemically volatile and flammable. How to preserve the cells without compromising the downstream analysis and safety is an urgent need for biomedical research.
Certain embodiments are directed to fixation methods that circumvent both biological and safety concerns associated with current fixation methods. The method allows for a minimum 24 hour transport of single cells in urine that remain genetically stable for downstream analysis. The method can be used to preserve single cells collected for clinical analysis in other liquid samples and bodily fluids. Described herein is a fixation method for preserving single cells in a biological sample (e.g., a biological fluid such as urine, blood, or cerebrospinal fluid (CSF)) samples. In certain aspects the biological sample is a post digital rectal examination (DRE) urine sample. In a further aspect the sample is in condition for downstream analysis. The samples can be fixed upon collection without compromising the integrity of RNA or DNA (e.g., in exfoliated prostate cells (PSA/PSMA positive)) for at least 24 hours. This has been validated using microfluidic qRT-PCR with a prostate cancer 48-gene panel. The method can stabilize the genetic materials in the cells and provide a snapshot of molecular profiling of cells that may be subject to changes during the storage and transportation. The fixation method is not limited to urine and can be used to preserve single cells in other clinical liquid samples or body fluids for downstream single cell analysis.
The invention includes the fixation of exfoliated prostate cells in post DRE urine samples for downstream single cell analysis. The final 0.2% of paraformaldehyde was used to fix the cells in urine samples post DRE by adding an appropriate volume of 4% paraformaldehyde/PBS. In certain aspects the method is used to fix the cells in the urine samples collected after DRE. The cells are lightly fixed immediately when the urine is collected and the cell RNA and DNA quality will remain about the same as that at the point of fixation. In certain aspects the RNA and DNA quality is maintained for at least 12, 24, or 36 hours. In a particular aspect the RNA and DNA quality is maintained for at least 24 hours.
Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.
Embodiments are directed to a fixative and methods for using the same in preparing a preserved sample or cells from a sample. In certain aspects a fixative comprises at least, at most, or about 2 to 5% paraformaldehyde, which is used to provide a final concentration of 0.2 to 0.5% in a fixed or preserved sample. In certain aspects the sample is a biological sample that comprises single cells. In a further aspect the biological sample is a urine sample, in particular a post digital rectal exam (DRE) urine sample. The fixative eliminates the need to immediately process urine samples. The fixative increases the stability of cells and maintains the integrity of nucleic acids in the cells, such as tumor cells, in urine or other biological samples. In further aspects the single cells can be isolated from the sample prior to being suspended in a solution with a final paraformaldehyde concentration of 0.2 to 0.5%. The cells can be separated from preserved urine samples using a number of different methods, including microfiltration, micromanipulation, and/or microinjection.
It will be appreciated given the description provided herein that the specific amounts of paraformaldehyde can vary in the stock solution and the fixative without departing from the excellent properties of the resulting fixative. The amount of paraformaldehyde in the stock solution may vary from between about 2 to about 20% (wt/vol). In certain aspects the stock solution may comprise 2 to 6% (wt/vol) paraformaldehyde. In particular aspects the stock solution can be about 4% paraformaldehyde. The fixed or preserved sample will have a final paraformaldehyde concentration of between 0.2 to 0.5% (wt/vol), including all values and ranges there between. In certain aspects the fixative will have a final paraformaldehyde concentration of about 0.2% (wt/vol).
The stock solution may be prepared by dissolving paraformaldehyde in deionized water. The pH of the reagent may be stabilized by adjusting it to between about 4.0 and about 10.0, including 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 9.0, 9.5 and 10.0. In a specific aspect, the pH is adjusted to about 7.4. pH stabilizing reagents for use in the reagents of the present invention include phosphate buffered saline (PBS), Tris-HCl, Hepes, citrate, and carbonate buffers, etc. In certain aspects the pH stabilizing reagent is PBS.
Upon mixing a stock solution of paraformaldehyde with a sample, such as urine, the final concentration of paraformaldehyde or fixative acts as a preservative that maintains the integrity of cells and the nucleic acids contained within the cells, i.e., decomposition of nucleic acids such as DNA, RNA and microRNA in the cells is inhibited or reduced. Typically, the stock paraformaldehyde solution is mixed with a sample within about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour of collecting the sample from a subject, or within about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minutes of collecting the sample from the subject. In particular aspects, the stock paraformaldehyde solution is mixed with the sample within about 3 hours, and preferably within 20 minutes, of collecting the sample from the subject.
Upon mixing the stock paraformaldehyde solution with the sample, forming a fixed or preserved sample, the fixed or preserved sample may be stored for a week at 4° C. until analyzed. For long term of storage, the stock can be frozen at −80° C. Alternatively, the fixed sample may be stored at room temperature or stored at a temperature between 0° C. and room temperature, such as at 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., or higher.
Many methods can be used for isolation of cells from the fixed sample. Centrifugation is one of the most common methods and involves use of centrifugal force for sedimentation of heterogeneous mixtures in the fixed sample. However, the pelleted materials at the bottom of centrifuge tube could contain not only cells, but also contaminating protein precipitates, cellular materials, red blood cells, and bacteria, etc.
Another method is to use antibodies or aptamers against the markers on the surface of cells to improve the purity of the collected material. Antibodies and aptamers can be attached to a solid support such as magnetic beads, ferrofluids, surfaces of microfluidic channels, etc. Magnetic beads or ferrofluids, coated with antibodies that recognize the cell surface marker of interest, can be mixed with fixed sample or a pellet collected from fixed sample. The cells will be captured on the surfaces of the magnetic beads or ferrofluids during incubation. Magnets can be used to collect the magnetic beads and ferrofluids and collect the cells of interest. If the antibodies or aptamers are coated on the surface of a microfluidic chip, the cells of interest will be captured on the surface of the microfluidic chip when the fixed sample flows through the chip.
Another method of affinity capture is to incubate the fixed sample or pellet from the fixed sample with antibody (antibodies) and/or aptamers, specific for cell surface markers of interest, that are conjugated with either avidin or biotin. After incubation, the cells can be collected on surfaces coated with biotin or avidin, respectively, to form biotin/avidin pairs. The surfaces can be magnetic beads, ferrofluids, microfluidic chips, etc.
The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Trypan blue cell survival assay. DU145 cells (ATCC) were cultured in vented-caps flasks (Corning, 430720U) in media RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco) and 1% Penecillin/Streptomycin (Gibco) at 37° C. in an Incubator fanned with 5% CO2. DU145 cells were harvested with 0.05% trypsin. About 50,000 cells were spiked in 1.5 ml fresh human urine in centrifuge tubes for 0 min, 30 min, and 60 min at room temperature. DU145 cells after urine incubation were separated from urine using centrifugation at 500×g for 5 min. The cell pellets were washed with PBS buffer twice and then stained with 0.2% trypan blue (Sigma-Aldrich, T8154) for cell survival assay. The cells without staining (trypan blue negative) were live cells and the cells stained blue were permeated dead cells. The cells were quantified using a Hemacytometer (Hausser Scientific, Cat #3120) (See,
Evaluation of cell RNA integrity. PC3, LNCaP, and BPH-1 prostate cancer cell lines were grown and harvested as previously described. About 0.5 million cells were spiked into 1.5 ml human urine at room temperature as above for 0 minute, 30 minutes, 1 hour, 2 hours, or 3 hours. The cells were centrifuged at 500×g for 5 minutes for collection and washed twice with 1 ml PBS. The cell pellets were lysed in Trizol LS reagent (Invitrogen, 10296-010) and RNA was isolated using RNAeasy mini kit (Qiagen, 74104) following the manufacturer's protocol. RNA (˜1 ng) integrity was evaluated using Agilent 2100 Bioanalyzer (Agilent Technlogies, G2939AA) according to the manufacturer's protocol.
Isolation of urine single cells and cultured prostate cancer cell lines. LNCaP cells (ATCC) were cultured in vented-caps flasks (Corning, 430720U) in media RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco) and 1% Penicillin/Streptomycin (Gibco) at 37° C. in an Incubator fanned with 5% CO2 Cells were trypsinized in Trypsin LE (Gibco), collected, centrifuged at 400×g and washed once in 1× PBS before being fixed in 0, 0.2, 0.3, 0.4, 0.5 or 1% paraformaldehyde for 20 minutes, 1 hour, or up to 24 hours at room temperature. For prostate cells in post-DRE urine samples, the paraformaldehyde was added according to the final concentrations for either 1 hour or 24 hours. Following fixation, the cells were centrifuged at 700×g (because of their increased buoyancy post-fixation, it was necessary to increase the centrifugation speed) then washed in 1×PBS two times. To prevent cells adhering to the plastic petri dishes during single-cell picking, cells were suspended in a 1:1 mix of 1× PBS and full culture media (described above) because the FBS prevents electrostatic “stickiness” between the cells and petri dish. Single cells were picked with a micromanipulator tip (Origio, MXL3-50) and ejected into a strip-PCR tube (Genessee, 27-125UA) with 4 μl of 0.4% Triton-X in 2× Reaction Mix (ThermoFisher Scientific, 11753-100) while keeping the samples at 4° C. after ejecting into the tube. Single-cell samples were frozen at −80° C. until reverse transcription and pre-amplification.
Single cell qRT-PCR using BioMark™ system. Single cultured prostate cancer cells or urine prostate cells isolated and stored in 2× reaction buffer were subject to single cell qRT-PCR using BioMark™ system following previously described. The gene panels used for gene expression profiling are androgen-upregulated gene panel V (Table 1), gene panel VII (Table 2). The gene expression levels were determined and all Ct values were compared to the expression of the housekeeping gene, UBB. Relative gene expression is based on negative delta delta Ct values (−ΔΔCt), which were calculated from a gene's max dCt value. Unsupervised hierarchical clustering and Principle Component Analysis (PCA) were performed in MeV. Network analysis was performed using Intelligent Pathway Analysis based on average gene expression (see,
Immunostaining. Dissociated cultured prostate cancer cells (˜0.5 million), BPH-1 and PC3, were fixed in 0.2% and 1% paraformaldehyde/PBS for 20 minutes and washed with PBS twice. Cell pellets were suspended in 100 μl PBS+5% FBS+0.2% tween 20 and cells were labeled with polyclonal rabbit α-PSA (1:100, Dako, #A0562), α-hPSMA/FOLH1-APC (1:10, R&D system, #FAB4234A) and incubated on ice for 15 min with light proof (aluminum foil). Cells were centrifuged in a bench microcentrifuge for 20 seconds. The cell pellets were washed 1 ml PBS+5% FBS+0.2% tween 20 to remove the primary antibodies. A secondary antibody (α-rabbit IgG-Cy3) was diluted (v:v=1:500) in 100 μl PBS+5% FBS+0.2% tween 20+0.5 μg/ml DAPI at RT for 15 minutes. The cells were washed with 100 μl PBS+5% FBS+0.2% tween 20 to removed the antibodies and removed into 6 well culture plate for fluorescent image analysis. The staining of PSA, FOLH1, and DAPI is located in cytoplasm, membrane, and nuclei, respectively. The images of immunostaining were taken using an inverted fluorescent microscope EVOS fl (Advanced Microscopy Group).
This Application claims priority to U.S. Provisional Patent application Ser. No. 62/281,012 filed Jan. 20, 2016, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62281012 | Jan 2016 | US |
