Patent Application
20100120047
For years, many researchers have attempted to perform molecular analyses on rare cells (also referred to herein as “target cells”) in various biological fluids (e.g., blood) that also contain assorted non-target cells. For example, one conventional system has had a certain degree of success at enumeration of circulating tumor cells (CTCs) as one type of target cell. However, this system has encountered extreme difficulty in evaluating a mutation status or analyzing mRNA transcipt levels of the target cells primarily because the background level of the non-target cells is too high, creating a target cell purity that is too low. For instance, the ratio of target cells to non-target cells can be 1 to 100, 1 to 1,000, or even lower.
Additionally, attempts at enabling selective transcript analysis have not been wholly successful. Some of these techniques avoid the problem of background non-target cells by analyzing only the mRNA transcripts which are uniquely expressed in the target cells, and which are not expressed in the non-target cells. In other words, with these types of systems, the apparent purity of the measured mRNA transcripts is essentially 100% because the non-target cells do not express mRNA transcripts. One such system analyzes unique target mRNA transcripts from enriched blood samples but is grossly restricted to just a few mRNA measurements which meet this expression criterion.
The present invention is directed toward a method for conducting a molecular analysis on target cells of a complex biological fluid that includes target cells and non-target cells, the method comprising the steps of reducing the complexity of the biological fluid by selectively depleting at least a portion of the non-target cells from the biological fluid, labeling the target cells or the non-target cells for identification, isolating the target cells from the non-target cells based on one of (i) a size, or (ii) a label, of the target cells or the non-target cells using a microfluidic sorting apparatus, and molecularly analyzing the target cells.
In one embodiment: step of reducing includes separating the non-target cells from the biological fluid using a Magnetic Activated Cell Sorting apparatus. In another embodiment, the step of separating includes using magnetic beads that include a plurality of different antibodies. The antibodies can include one or more of CD45, CD16 and 235a Further, the step of reducing can include lysing at least some of the non-target cells. The step of lysing can include using an ammonium chloride solution. The step of reducing can also or alternatively include filtration of at least some of the non-target cells.
In certain embodiments, the step of labeling can include labeling both the target cells and the non-target cells. In another embodiment, the step of labeling can include using a label selected from the group consisting of fluorescent, colorimetric, magnetic and biochemical labels. Additionally, or in the alternative, the microfluidic sorting apparatus can include a microfluidic Fluorescence Activated Cell Sorting apparatus.
In one embodiment, the step of isolating includes purifying the target cells based on a label of the target cells and the non-target cells. The step of isolating can include purifying the target cells based on the size and the label of either the target cells or the non-target cells. Further, in one embodiment, the number of target and non-target cells in the biological fluid can be less than approximately 1×1011 cells in a fluid volume of less than approximately 100 mls.
In another embodiment, the step of isolating can include recovering the target cells in a volume of at least approximately 5 μl and less than approximately 1 ml. Further, the step of isolating can include recovering the target cells in a volume of at least approximately 10 μl and less than approximately 0.1 ml. In one embodiment, the biological fluid is selected from the group consisting of blood sputum, peritoneal fluid, tissue cell suspension, fine needle aspirates, fecal matter and cerebral spinal fluid. In another embodiment, the step of isolating yields a purity of at least approximately 60%.
Further, the target cells can be selected from the group consisting of tumor cells, fetal cells, stem cells, endothelial cells, myocardial cells and lymphocytes. In some embodiments, the duration of the method is less than approximately 6 hours. In certain embodiments, the step of isolating includes retention of the target cells being at least 80%. In another embodiment, the step of molecularly analyzing the target cells can include one of RNA analysis, antigen expression and mutation profiling.
The invention, together with further advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, taken in conjunction with the accompanying description, in which similar reference characters refer to similar parts, and in which:
The methods provided herein allow an increased purification of rare cells (also referred to herein as “target cells”) from complex biological fluids that also include non-rare cells (also referred to herein as “non-target cells”). The process uniquely utilizes techniques that yield unexpected results, i.e. rapid, efficient recovery of purified rare cells which have utility in a variety of assays. As provided herein, the duration of the described processes is decreased from conventional processes, while enabling expedited diagnoses, enhancing viability and reducing and/or eliminating changes to the biology of cells. In certain embodiments, relatively large volumes such as 100 mLs of blood can be reduced to 5 μl or less of a substantially pure population of target cells which can be initially present at extremely low levels, such as only 1 cell/mL or less. In one embodiment, the purity approaches or even achieve approximately 100% with recoveries routinely exceeding approximately 80%. The collection of essentially pure target cells has enabled a range of analyses, biological insights and treatment modalities that were previously impractical, if not impossible to attain.
At step 110, the complexity of the biological fluid is reduced by at least a partial selective (or differential) depletion of the non-target cells. In this step, in one embodiment, a modest or moderate reduction of the complexity of the biological fluid is desired and achieved. Examples of non-target cells can include, but are not limited to, red blood cells (RBCs) and white blood cells (WBCs). In one embodiment, a technique that is used for differential depletion of non-target cells can include lysis of RBCs using various solutions such as an ammonium chloride solution.
In non-exclusive alternative embodiments, the technique can include RBC removal using filtration or induced paramagnetic separation. RBCs and WBCs can be differentially removed using techniques of microfluidic separation, differential electrophoretic mobility, Magnetic Activated Cell Sorting (MACS), Fluorescence Activated Cell Sorting (FACS), centrifugation, gradient isolation and other suitable techniques. Utilizing the techniques provided herein, the retention of target cells over the depletion of non-target cells such as RBCs and WBCs is increased. In one embodiment, combining this initial step with the downstream steps provides increased speed and target retention, and ultimately, a higher likelihood of achieving or approaching cell purity. In one embodiment, lysis of the RBCs and/or WBCs can reduce the total number of cells from approximately 1×109 cells per ml. to fewer than approximately 1×108 cells per ml. In non-exclusive alternative embodiments, the number of cells can be reduced to fewer than approximately 5×107 cells per ml. or 1×107 cells per ml.
As set forth in greater detail below, concurrently or following removal of at least a portion of the RBCs and/or WBCs, other types of cells can also be removed using various techniques including, but not limited to, magnetic depletion and/or separation. With this technique, in one embodiment, the number of cells can be reduced from approximately 1×109 cells per ml. (or fewer after RBC and/or WBC depletion) to fewer than approximately 1×107 cells per ml. In non-exclusive alternative embodiments, the number of cells can be reduced to fewer than approximately 5×106 cells per ml., 1×106 cells per ml., 5×105 cells per ml., or 1×105 cells per ml. Stated another way, in non-exclusive alternative embodiments, the total cell count of the biological fluid can be reduced by a factor of at least approximately 1×102, 5×102, 1×103, 5×103, or 1×104, or even greater.
It should be noted that it is not necessary to remove 100% of the non-target cells or any one type of non-target cell from the biological fluid at this step. In fact, attempting to remove 100% of one or more types of non-target cells from the biological fluid at this step tends to reduce the retention of the target cells. Therefore, it is only necessary to use this first step as a preliminary screening process to remove a certain percentage of the population of non-target cells. In one embodiment, approximately 99.99% of the non-target cells are removed. In other words, the non-target cells are reduced by a factor of approximately 104. In various alternative embodiments, less than approximately 99.9%, 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5% of the non-target cells are removed depending. upon (i) the type of biological fluid being analyzed, (ii) the types of target and non-target cells within the biological fluid, and/or (iii) the desired percentage of purity of the target cells from the biological fluid.
As provided above, at step 220, the RBCs (and/or WBCs) are selectively depleted by the lysis techniques described herein or by another suitable method that depletes the RBCs and/or WBCs.
At step 222, some or all of the leukocytes of the biological fluid can be removed. In one embodiment, anti-CD45-covered magnetic beads can be added to a suspension of the biological fluid remaining following RBC and/or WBC depletion. The volume of anti-CD45-covered magnetic beads can vary depending upon the total volume of the suspension. For example, in one embodiment, approximately 40 μl of anti-CD45-covered magnetic beads can be added per 1 ml. of suspension. However, it is recognized that more or less than 40 μl of anti-CD45-covered magnetic beads can be added per 1 ml. of suspension. The leukocytes effectively “stick” to the anti-CD45-covered magnetic beads and can consequently be removed from the suspension. Alternatively, another type of antibody can be used in conjunction with magnetic beads to remove the leukocytes.
At step 224, some or all of the neutrophils of the biological fluid can be removed. In one embodiment, anti-CD16-covered magnetic beads can be added to the suspension of biological fluid remaining following RBC and/or WBC depletion. The volume of anti-CD16-covered magnetic beads can vary depending upon the total volume of the suspension. For example, in one embodiment, approximately 200 μl of anti-CD16-covered magnetic beads can be added per 1 ml. of suspension. However, it is recognized that more or less than 200 μl of anti-CD45-covered magnetic beads can be added per 1 ml. of suspension. The neutrophils effectively “stick” to the anti-CD16-covered magnetic beads and can consequently be removed from the suspension. Alternatively, another type of antibody can be used in conjunction with magnetic beads to remove the neutrophils.
At step 226, some or all of the residual RBCs of the biological fluid can be removed. In one embodiment, anti-CD235a-covered magnetic beads can be added to the suspension of biological fluid remaining following the initial lysing of the RBCs and/or WBCs described previously. The volume of anti-CD235a-covered magnetic beads can vary depending upon the total volume of the suspension. The RBCs effectively “stick” to the anti-CD235a-covered magnetic beads and can consequently be removed from the suspension. Alternatively, another type of antibody can be used in conjunction with magnetic beads to remove the RBCs.
At step 228, some or all of the platelets of the biological fluid can be removed. In one embodiment, anti-CD41a-covered magnetic beads can be added to the suspension of biological fluid remaining following RBC and/or WBC depletion. The volume of anti-CD41a-covered magnetic beads can vary depending upon the total volume of the suspension. For example, in one embodiment, approximately 10 μl of anti-CD41a-covered magnetic beads can be added per 1 ml. of suspension. However, it is recognized that more or less than 10 μl of anti-CD41a-covered magnetic beads can be added per 1 ml. of suspension. The platelets effectively “stick” to the anti-CD41a-covered magnetic beads and can consequently be removed from the suspension. Alternatively, another type of antibody can be used in conjunction with magnetic beads to remove the platelets.
It is recognized that in one embodiment, removal of cells that occurs in some or all of steps 222, 224, 226 and/or 228 can occur either simultaneously by using an “antibody cocktail” that includes a plurality of antibody-covered magnetic beads, or the removal can be carried out in a sequence utilizing one or more antibodies at a time, which can occur in a sequence that differs from steps 222, 224, 226 and 228 described above, for example.
Referring back to
In one embodiment, a plurality of different antibody specificities can be used which increases the detection of rare cells. For instance, epithelial cells and/or other types of cells such as WBCs can be labeled with phycoerithritin (PE) labeled antibody solutions such as CD326-PE (Epithelial Cell Adhesion Molecule, also referred to as “EpCam”), pan-cytokeratin antibodies, anti-cytokeratin (anti-CK), anti-CD45-TC,2′-(4-Ethoxyphenyl)-5-(4-mrthly-1-piperazinyl)-2,5′-bis-1H enzimidazole.3HCl (also referred to as “H33342”) and/or other suitable labels, as non-exclusive examples. Additionally, or in the alternative, organ-specific (or tissue-specific) markers can be included in the plurality of labels to increase the likelihood that cells from rare tumor types that may have low expression of EpCam or cytokeratin are also detected. For example, tissues such as the pancreas present MUC1, ErbB2 and MUC4 proteins, colorectal cells present CEACAM, prostate cells present PSA, lung cells present EGFR and Ovarian cells present CA-125, CA-72-4, CA-19-9 and CEA, as non-exclusive examples.
The specific volumes of any or all of the above solutions can be varied depending upon the total volume of target and non-target cells being labeled. In some embodiments, a plurality of antibodies can use the same fluorescent label or distinct labels for differentiation as needed. In certain embodiments, a labeling strategy can be advantageous over binding the target cells to beads or surfaces which do not readily have access to binding of cytoplasmic markers and require release strategies that often fragment cells for many downstream analyses.
At step 114, target cells are isolated based on their size and/or label. As used herein, microfluidic sorting can include microfluidic sorting apparatus such as a microfluidic Fluorescence Activated Cell Sorting apparatus (also referred to herein as “μFACS” or “microfluidic FACS”). As used herein, microfluidic FACS is defined as one or more devices which use fluorescence and/or optical detection and measurements to segregate cells based on their size and/or labels. Cells flow through these devices in a relatively narrow fluid stream at relatively low shear stress. Further, these devices can be configured for flow rates that facilitate accurate label identification and subsequent sorting at relatively high efficiency from a relatively small number of cells. These instruments can sort through cells in numbers that range from less than one thousand to greater than one million, which is a departure from traditional FACS instruments. In the present invention, the combination of one or more of step 1 (low purity and modest complexity reduction), step 2 (labeling of target and or non-target cells for identification) and step 3 (μFACS for rapid purity generation of the initially high complexity sample) generates unexpected results of substantial purity, greater target retention and increased speed for utilization in step 116, as explained in greater detail below.
At step 116, the target cells obtained from steps 110, 112 and 114 are molecularly analyzed. The methods provided herein isolate cells that are compatible with numerous downstream molecular analysis applications. These applications can include, as non-exclusive representative examples, DNA analyses, RNA analyses, antigen expression, proteomic analyses and other biological assays as known to one skilled in the art. More specifically, DNA analyses can include DNA microarrays, Comparative Genomic Hybridization (CGH), analysis for rare mutant alleles (single-nucleotide polymorphism (SNP), short tandem repeat (STR), and other microsatellite mutations), mitochondrial DNA, or fluorescent in-situ hybridization (FISH), as non-exclusive examples. RNA analyses can include transcriptional analysis via RT-qPCR and RNA microarrays, as non-exclusive examples. Proteomic analysis can include cell signaling, mass spectrometry, and sub-cellular protein localization, as non-exclusive examples. Biological assays can include viability, drug sensitivity and enzymology, as non-exclusive examples. It is understood that the foregoing listing of various types of molecular analyses are provided as examples only, and are not intended to limit the scope of the present invention in any manner.
Many analyses rely on purity for more accurate results, including but not limited to: CGH, rare allele detection and/or transcriptional profiling. In these cases, enrichment methods are not enabling. For instance, determining the chromosome copy number in aneuploid fetal cells without the presence of contaminating maternal euploid cells is crucial. In all cases, having pure populations can allow more acute and/or accurate measurements of the population of cells and the variability inherent within. This is particularly important for therapeutic applications, for example.
Advantageously, one or more of the methods provided herein do not require harsh fixatives or binding of cells to surfaces such as slides, chips or beads. Binding to surfaces can require manipulation and cell release for some characterizations or for collection and injection back into a host. While manipulating cells adhered to surfaces may not only be harsh, often resulting in a loss of viability or partial destruction of the cell, such manipulations are time consuming and inherently not commercially scalable. However, in an alternative embodiment, cells can be fixed and/or otherwise adhered to slides or other surfaces used by one familiar in the art, if desired.
In this example, 100 prostate cancer cells treated with taxol to induce mitotic arrest were spiked-in to whole blood and stored for four days. Using the methods provided herein, 72 target cells were recovered (illustrated in the somewhat trapezoidal region in the upper portion of the graph in
The purification of target cells from blood can be verified by analysis of cell transcripts. If transcripts are measured by RT-qPCR the resulting Ct values can be compared for each transcript from one sample to another. When Cts from target cells are compared to the whole blood the cells were spiked into, there is virtually no correlation, as illustrated in
The third column in
The fourth column in
The fifth column in
Cells of diagnostic and therapeutic value exist in body fluids and tissue suspensions. Example fluids can include, but are not limited to, circulatory blood, disassociated tissue suspensions, cord blood, urine, sputum, cerebral spinal fluid, fecal matter suspensions and peritoneal fluid, which have all been monitored for biomarkers of disease, or other suitable fluids. Target cells of interest can include, but are not limited to, circulating epithelial cells such as those indicative of cancer, and circulating endothelial cells as markers of vascular damage such as myocardial infarction, vasculitis and transplantation. For example, collection of fetal cells from maternal blood or cord blood would enable a non-invasive prenatal health diagnosis to be made. Stem cells may also be isolated from a patient's blood, marrow, organs or other body fluids, appropriately differentiated, propagated and used to supplant failing tissues or organs. Other target cells can include one or more of tumor cells, endothelial cells, myocardial cells and lymphocytes, as non-exclusive examples.
One or more of the methods provided herein have essentially no theoretical volume limit and no measured complexity limit. Rare cell events may be present at less than 1 cell/mL of blood. Further, one or more of the methods provided herein overcome the previously unsolved problem regarding scalability for relatively large initial fluid volumes, such as greater than 7.5 mLs, for instance. Biological fluids have varying degrees of complexity, with blood having the highest density of cells (1×109/mL) in human body fluids. In certain embodiments, one or more of the methods provided herein can rapidly reduce approximately 100 mLs of blood (approximately 1×1011 cells) to substantially purified populations of cells on the order of μl volumes. Thus, input and output volumes of several orders of magnitude can be managed and achieved utilizing the methods provided herein. In non-exclusive alternative embodiments, the target cells can be recovered in a volume of at least approximately 5 μl and less than approximately 1 ml, or at least approximately 10 μl and less than approximately 0.1 ml. Still alternatively, other blood volumes can be rapidly reduced to substantially purified populations of cells on the order of volumes that are somewhat proportionate to the above examples.
Many of the methods provided herein allow for relatively high purity and recovery retention rates. Previously, retention of target cells was measureable when a gold standard exists for the quantity existing in the sample. The best way to generate such a standard is to spike-in a known quantity of cells and measure the output purity and recovery retention rates. The present invention is capable of routinely achieving greater than approximately 60%, 70%, 80% or 90% purity and/or recovery retention.
Additionally, one or more of the methods described herein can permit a decreased processing time. Further, the entire process can be scalable by a single operator in a single location. This has commercial value in the ability to process many samples simultaneously and provide data back to a clinician, researcher or patient in point of care settings. Additionally, many of the processes disclosed herein allow the identification of many transient biological events such as cell signaling and would be missed by longer enrichment methodologies.
In certain embodiments, selective depletion of non-target cells and sorting of target cells can occur by a multi-parameter analysis. Size and labels such as DNA content and as many external or cytoplasmic antigens as one wishes can be used individually or in sets of positive and negative labels for selection. Conventional techniques that rely on affinity capture are not easily scaled to use multiple antibodies due to the loss of co-operativity that results as a single capture antibody is diluted out by second, third or other additional antibody types. This issue is negated when antibody labels are bound to cells in bulk solution. Information on the size of the target cells can also be collected using the methods of the present invention and can be incorporated into the selection process, if desired.
In one embodiment, the duration of the entire method described herein can be less than approximately 3 hours. In non-exclusive alternative embodiments, the duration of the entire method described herein can be less than approximately 6 hours or less than approximately one day.
While the methods as shown and disclosed herein are fully capable of obtaining the objects and providing the advantages herein before stated, it is to be understood that they are merely illustrative of the presently preferred embodiments of the invention and that no limitations are intended to the details of the methods, processes, construction or design herein shown and described.
This Application claims domestic priority on U.S. Provisional Application Ser. No. 61/112,516, filed on Nov. 7, 2008. The contents of U.S. Provisional Application Ser. Nos. 61/112,516 are incorporated herein by reference to the extent permitted.
| Number | Date | Country | |
|---|---|---|---|
| 61112516 | Nov 2008 | US |
