WO2013049670A1

WO2013049670A1 - Method for optimizing the collection of rare cells in blood

Info

Publication number: WO2013049670A1
Application number: PCT/US2012/058055
Authority: WO
Inventors: David M. NELSON; Dena Marrinucci; Daniel BOFFA
Original assignee: Epic Sciences, Inc.; Yale School of Medicine
Priority date: 2011-09-28
Filing date: 2012-09-28
Publication date: 2013-04-04

Abstract

Disclosed herein are methods and kits that can effectively collect rare cells, for example, circulating tumor cells (CTCs), in blood. The methods, in some embodiments, include the use of blood collection devices, such as needles and catheters, that are 18 gauge or larger. Also disclosed herein are kits for optimizing the collection of rare cells in blood.

Description

METHOD FOR OPTIMIZING THE COLLECTION OF RARE CELLS IN BLOOD

FD2LD OF THE INVENTION

[0001] The present disclosure relates to the field of cell biology and medicine. More particularly, disclosed herein are methods for optimizing collection of rare cells in blood by using large gauge needles for collecting blood samples.

BACKGROUND INFORMATION

[0002] Various types of rare cells have been identified in blood. Some of those rare cells can be used to diagnose, monitor, and screen unusual or abnormal conditions, such as pregnancy, infectious diseases and cancer. For example, circulating tumor cells (CTCs), which are cells that have detached from a primary tumor and circulate in the bloodstream, are thought to be the seed of subsequent growth of additional tumors (metastasis) in different tissues. Detection and characterization of CTCs by molecular analysis are thought to be valuable for stratifying cancer patients and aiding with individualized treatment strategies.

[0003] The number and quality of intact rare cells found in the blood samples collected using standard blood-collection procedures is fairly low. Therefore, there is a need for methods that can optimize the collection of rare cells in blood.

BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1A is schematic representation of a microcirculation (capillary bed or liver sinusoids) demonstrating the size differential.

[0005] Figure IB is schematic representation outlining the presumed path of circulating tumor cells (CTCs) shed through the venous drainage of a breast tumor.

[0006] Figure 1C is schematic representation outlining the path of CTCs shed by a colon cancer.

[0007] Figure ID is schematic representation outlining the unique drainage of a lung cancer into the left sided circulation without having passed through a capillary bed. [0008] Figure 2 is a histogram showing the combined number of CTCs per 2 ml (y axis) for each patient (x axis). The numbers of CTCs in arterial blood are shown on the bottom of each bar (black), and the numbers of CTCs in venous blood are shown at the top of each bar (gray).

[0009] Figure 3 is a schematic diagram illustrating left heart catheterization. (A) a patient with clinical stage 2 lung cancer, and (B) a patient with a clinical stage 1 lung cancer.

DETAILED DESCRIPTION

[0010] The illustrative embodiments described in the following detailed description and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects of the present disclosure, as generally described herein, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this disclosure.

[0011] The present disclosure provides methods and kits for effectively collecting rare cells in blood from a human being or other animal subject. It has been appreciated in the art that too few rare cells can be obtained from blood samples collected using standard collection procedures, which presents a challenging problem for downstream cell analysis and characterization. Without being bound by any particular theory, it is believed that the number of rare cells obtained from a blood sample may be limited, at least in part, by the way that the blood sample is collected.

[0012] As used herein, the term "rare cells" refers to rare occurring cells in the blood of a human being or other animal subject. For example, the rare cells can be cells that are not normally present in blood, but may be present in blood as a result of an unusual or abnormal condition, such as pregnancy, infectious disease, chronic disease, or injury. Rare cells can also be cells that may be normally present in blood, but are present with a frequency several orders of magnitude less than cells typically present in a normal blood specimen. In some embodiments, the rare cells are more fragile than the other cells that are normally present in blood (e.g., white blood cells and/or red blood cells). Examples of rare cells in blood include, but are not limited to, circulating tumor cells (CTCs), circulating endothelia cells (CECs), fetal cells, stem cells, and any combination thereof. In some embodiments, the rare cell is a CTC. In some embodiments, the rare cell is a fetal cell. In some embodiments, the rare cell is a stem cell.

[0013] As disclosed herein, the use of blood collection devices, for example a needle or a catheter, with large gauge can significantly improve, and thus optimize, the number and/or integrity of rare cells collected from the blood. Such an improvement and optimization can be advantageous for analysis and/or characterization of the rare cells. Some embodiments provide a method for analyzing rare cells in blood, wherein the method comprises: providing a sample of blood from a subject in a container with at least one preservative, wherein the sample is obtained from the subject using a needle or catheter that is at least as large as 20 gauge; identifying at least one rare cell in the sample; and analyzing the rare cell at least about 6 hours after obtaining the sample from the subject. The container can be a tube (e.g., a blood collection tube); an eppendorf tube; or a bag (e.g., a blood collection bag).

[0014] In some embodiments, the subject is suffering from cancer. In other embodiments, the subject is suspected of having cancer.

[0015] In some embodiments, the needle or catheter is at least as large as 20 gauge, 19 gauge, 18 gauge, 17 gauge, 16 gauge, 15 gauge, 14 gauge, 13 gauge, 12 gauge, 11 gauge, 10 gauge, 9 gauge, 8 gauge, or 7 gauge. In some preferred embodiments, the needle or catheter is at least as large as 18 gauge. In some preferred embodiments, the needle or catheter is at least as large as 16 gauge. In some preferred embodiments, the needle or catheter is at least as large as 14 gauge. Without being limited to any particular theory, it is believed that the use of a blood collection device with large gauge can lead to less damage to the rare cells during blood collection as compared to the use of smaller needle or catheters due to reduced shear stress on the cells, and/or reduced mechanical separation of clusters of the rare cells. In some embodiments, a blood collection device, such as a needle or catheter, having a size of approximately 20 gauge, 19 gauge, 18 gauge, 17 gauge, 16 gauge, 15 gauge, 14 gauge, 13 gauge, 12 gauge, 11 gauge, 10 gauge, 9 gauge, 8 gauge, or 7 gauge can be used. Needles, catheters, and other devices having similar sizes to those disclosed here, but measured according to other scales (such as inches, millimeters, or the French catheter scale) are also contemplated for use in the present invention. [001 ] When selecting the size of blood collection device, factors such as the disease condition of the patient, age of the patient, location for blood draw, and type of rare cells to be collected can be considered. In some embodiments, local or general anesthesia is applied to the patient for the blood draw.

[0017] In some embodiments, the use of a blood collection device that is at least as large as 20 gauge can improve the number and/or the integrity of the rare cells collected as compared to the use of blood collection device with smaller gauge. In some embodiments, the use of blood collection device that is at least as large as 20 gauge can prevent, alleviate, or reduce separation of clusters of rare cells as compared to the use of blood collection device with smaller gauge. In some embodiments, the use of a blood collection device that is at least as large as 20 gauge can better preserve clusters of rare cells as compared to the use of a blood collection device with a smaller gauge. In some embodiments, the use of a blood collection device that is at least as large as 20 gauge can reduce the sheer force applied to the rare cells in the blood sample, and thereby increase the number of intact rare cells obtained, as compared to the use of a blood collection device with a smaller gauge.

[0018] In some embodiments, the number of the rare cells obtained from a blood sample using a blood collection device that is at least as large as 20 gauge is increased by at least about 50%, at least about 75%, at least about 100%, at least about 125%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% as compared to the number of the rare cells obtained from another blood sample having the same volume from the same subject using a blood collection device with a smaller gauge. In some embodiments, the number of the rare cells obtained from a blood sample using a blood collection device that is at least as large as 20 gauge is increased by about 50%, about 75%, about 100%, about 125%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or ranges between any two of these values, as compared to the number of the rare cells obtained from another blood sample having the same volume from the same subject using a blood collection device with smaller gauge.

[0019] The site from which the blood sample is drawn from the subject can vary. For example, the blood sample can be drawn from a vein or an artery in the subject. In some embodiments, the sample is venous blood. In some embodiments, the sample is arterial blood. In some embodiments, a larger number of rare cells, for example CTCs, are found in venous blood as compared to arterial blood of the same volume.

[0020] As used herein, the term "enrichment" refers to the process of substantially increasing the ratio of a target bioentity {e.g., rare cells in blood) to non-target materials in the processed analytical sample compared to the ratio in the original biological sample. In some embodiments, rare cells can be enriched so that the ratio of the rare cells and the non-target material in the blood {e.g., white blood cells) is increased by at least about 10 fold, at least about 100 fold, at least about 500 fold, at least about 1000 fold, at least about 2000 fold, or at least about 5000 fold.

[0021] In some embodiments, the rare cell is analyzed at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, or at least about 24 hours after the blood sample is obtained from the subject. In some embodiments, the rare cell is analyzed about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, or ranges between any two of these values after the blood sample is obtained from a subject. In some embodiments, the rare cell is analyzed about 6 to about 8 hours after the blood sample is obtained from a patient.

[0022] As disclosed herein, a blood sample can be stored in a container with at least one preservative after being obtained from the subject. In some embodiments, the preservative can stop decomposition of the blood and/or prevent bacterial contamination. For example, the preservative can be sodium fluoride (NaF). In some embodiments, the blood sample can be contacted with an agent capable of maintaining the structure integrity of the rare cells in the sample. The agent can, in some embodiment, prevent or reduce mechanical and/or chemical damages to the rare cells for at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, or at least about 24 hours. In some embodiments, the agent can prevent or reduce mechanical and/or chemical damage to the rare cells for at least up to 24 hours, at least up to 36 hours, at least up to 48 hours, or at least up to 72 hours. Such long-term stability is preferred in cases where the blood sample is obtained in a location that is distant to the location where the identification of rare cells in the sample and/or the analysis of the rare cells will occur. In some embodiments, the blood sample is stabilized against mechanical damage during transport.

[0023] Any suitable methods known in the art can be used to determine the structural integrity of the rare cells. Non-limiting examples of such methods include immunocytochemical procedures, RT-PCR procedures, PCR procedures, FISH procedures, flowcytometry procedures, image cytometry procedures, and any combination thereof.

[0024] Any suitable methods known in the art can be used to identify the rare cells. In some embodiments, the method allows obtaining rare cells, for example individual rare cells, without significant disruption of the cells. Therefore, these methods allow preservation of cytological details of the cells and detailed downstream analysis of the cells. Non-limiting examples of methods for identifying rare cells include biochemical assays, immunochemical assays, immunofluoresence assay, and immunocytochemistry assays. For example, many analytical methods are known in the art to characterize and/or analyze tumor cells (e.g., CTCs). In some embodiments, CTCs can identified by the expression of the cytoskeletal proteins cytokeratin (CK+), the absence of the common leukocyte antigen CD45 (CD45-) and the presence of nucleic acids (NA+) by multicolor fluorescence analysis. A variety of methods have been used for identifying CTCs. See, e.g., Marrinucci et al., Arch. Pathol. Lab. Med., 133:1468-1471 (2009); U.S. Application No. 61/435704, filed January 24, 2011, both of which are hereby incorporated by reference in their entirety. In some embodiments, the CTCs are identified by using CellSearch^® assay from Veridex. In some embodiments, the blood sample can be enriched for the rare cells that expressed EpCAM. Rare cells can also be immunophenotyped by both flowcytometry and fluorescence microscopy.

[0025] Examples of methods that can be used for downstream analyses to characterize and/or analyze the cells include, but are not limited to, biochemical analysis; immunochemical analysis; image analysis; cytomorphological analysis; molecule analysis such as PCR, sequencing, determination of DNA methylation; proteomics analysis such as determination of protein glycosylation and/or phosphorylation pattern; genomics analysis; epigenomics analysis; transcriptomics analysis; and any combination thereof.

[0026] In some embodiments, the methods disclosed herein include enriching the rare cells in the sample. A variety of methods are known in the art to enrich predetermined calls in a sample. Such methods have been used to enrich fetal cells from a sample of maternal peripheral blood and tumor cells from bodily fluid. For example, cell sorting by FACS technology has been applied to enumerate and collect rare cells in biological samples. Several immunochemical methods, including immunocapturing methods, have also been developed for the enrichment of cells from fluid specimens using solid phase absorption. U.S. Patent Publication No. 20100285581 describes methods for enriching cells of interest with high purity based on solid phase isolation (which is hereby incorporated by reference in its entirety). Skilled artisan will appreciate that any suitable methods known in the art can be used to enrich rare cells in the methods and kits disclosed herein.

[0027] Also enclosed herein are kits for collecting a blood sample that includes rare cells in blood. In some embodiments, the kit comprises a blood collection device, for example a needle or catheter, which is at least as large as 20 gauge, 19 gauge, 18 gauge, 17 gauge, 16 gauge, 15 gauge, 14 gauge, 13 gauge, 12 gauge, 11 gauge, 10 gauge, 9 gauge, 8 gauge, or 7 gauge; and a container with at least one agent capable of maintaining the structural integrity of the rare cell in the sample. In some embodiments, a blood collection device at least as large as 18 gauge is preferred. In some embodiments, a blood collection device at least as large at 14 gauge is preferred.

EXAMPLES

[0028] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.

EXAMPLE 1

COLLECTION OF CIRCULATING TUMOR CELLS (CTCs)

[0029] In this example, two samples of venous blood were drawn from each patient using catheters of different size (made by Becton, Dickinson and Company of Franklin Lakes, NJ ("BD")). Circulating tumor cells (CTCs) were collected from each sample and the numbers of the CTCs were counted. The results are shown in Table 1.

[0030] As shown in Table 1, the use of catheters of larger size resulted in collection of more CTCs as compared to the use of catheters of smaller size.

Table 1. Numbers of CTCs collected using various sizes of catheters

EXAMPLE 2

COLLECTION OF CIRCULATING TUMOR CELLS (CTCs)

[0031] In this example, venous and/or arterial blood was drawn from 9 patients. CTCs and CTC clusters in each blood sample were characterized. Cells that did not express cytokeratin ("CK") were identified, and named as "No CK" cells. These "No CK" cells were not classical cancer cells because they lack CK expression, but were likely to be cancer stem cells or epithelial to mesenchymal transition (EMT) cells. The results are shown in Table 2. As shown in Table 2, large clusters of CTCs were retrieved from several patients, including Patients No. 3 and 4.

[0032] The clusters were categorized by the number of cells per cluster. "Cluster Size/mL" in Table 2 refers to the number of clusters found per mL that had 2-3, 4-9, or 10+ cells per cluster.

[0033] As shown in Table 2, CTC quantities from peripheral arterial blood samples were found to be different from those obtained from peripheral venous blood. Clinicians are advised to maintain a consistent source of blood throughout longitudinal studies, as a source- dependant variability that could obscure important clinical differences that been identified. This point is particularly important in surgically managed patients, as commonly placed arterial catheters provide a convenient access for blood specimens.

Table 2. Collection of CTCs and CTC clusters

* B Braun - B. Braun Melsungen AG (Melsungen, Germany)

BD - Braun Becton, Dickinson and Company (Franklin Lakes, NJ);

Arrow - Arrow (Teleflex Incorporated, USA);

Butterfly - butterfly needle manufactured by Becton, Dickinson and Company (Franklin Lakes, NJ). EXAMPLE 3

COMPARISON OF CIRCULATING TUMOR CELLS (CTCs) COLLECTED FROM

VENOUS BLOOD AND ARTERIAL BLOOD

[0034] In this example, peripheral arterial and venous blood specimens from 44 patients with primary lung cancer were examined. The frequency of cells identified and collected by immunomagnetic precipitation (n = 9) was too low to allow a meaningful comparison. Using scanning microscopy (n = 35), CTCs were found to be significantly less prevalent (i.e., detected in less frequency) in the arterial circulation than the venous circulation (p = 0.002). Clusters of 2 or more circulating cells were similarly less common among the arterial specimens (p = 0.001). As a result, clinicians should be cautioned when using arterial blood draws for CTCs as a biomarker, as the results may differ from those obtained from venous blood.

[0035] Tumor cell circulation is a critical component of metastatic progression and circulating tumor cells (CTCs) have been isolated from the blood of patients with most tumor types [1]. The prevalence of CTCs in the peripheral venous blood has been correlated with tumor stage [2], the development of metastases [3], responses to therapy [4], recurrence [5] and survival [3, 6-11]. Circulating tumor cells (CTCs) have shown great promise as a biomarker in patients with solid tumors. Thus far, the vast majority of investigations have taken place using peripheral venous blood samples.

[0036] Despite the promise of peripheral venous blood as a potential biomarker, several aspects of the microcirculation suggest that the peripheral venous blood may not represent the complete circulating tumor cell burden. For example, tumor cell clusters as well as individual tumor cells are thought to be significantly larger than the capillaries that separate the arterial and venous circulations. The size of a typical tumor cell in the circulation (20 um) is 2-4 times larger than the smallest lumen of a capillary bed (< 8um) [12, 13]. Moreover tumor cells are known to circulate in clusters, further amplifying this size discrepancy [14]. In addition, the endothelial adhesion molecule expression within a capillary bed likely traps a substantial proportion of CTCs [12, 15]. At least one report suggests that the majority of circulating tumor cells is trapped in the first pass through a capillary bed [16] (Figure 1A). The vascular drainage patterns of most tumors result in at least two of these potentially restrictive microcirculations being interposed between the primary tumor and a peripheral vein (Figure IB). It is possible that there are important differences in the arterial and venous circulations, due in part to the selection of the interposed microcirculation.

[0037] Primary lung cancer provides a unique opportunity to study the impact of microcirculation on the circulating tumor cell populations in the arterial and venous circulations. Although primary lung cancers obtain oxygenated blood through the systemic arterial circulation they drain directly into the left heart via the pulmonary veins [17] (Figure 1C). As a result, lung cancer tumor cells enter the peripheral arterial circulation without having crossed a capillary bed (unlike most tumors, which have to pass through the pulmonary capillary bed before reaching the peripheral arterial circulation). Therefore, a comparison of CTCs within the peripheral arterial and venous circulations provides a unique opportunity to determine the impact of a single level of capillary beds (including size restriction and endothelial adhesion) on the CTC populations.

[0038] In this example, possible causes for the differences in the prevalence of circulating tumor cells between peripheral arterial and venous circulations, for example interposing microcirculation, were investigated. Since the peripheral arterial and venous circulations are separated by a single capillary bed, lung cancer population was used for the investigation. To evaluate differences in the CTC populations, CTCs were quantified in peripheral arterial and venous blood from patients presumed to have surgically resectable primary lung cancer were studied using immunomagnetic precipitation (CellSearch^®) and scanning microscopy (Epic Science method) for quantification.

Methods and materials

[0039] Patients and Specimen Collection: Primary lung cancer patients seen at Yale New Haven Hospital between March and September of 2011 that were felt to have surgically resectable disease (clinically staged Ι-ΙΠ) were evaluated. Individuals that were scheduled to undergo a procedure that provided access to the peripheral venous and arterial circulations were eligible for the study and were consented in accordance with our IRB approved protocol (patients less than 18 years of age, non English speaking, or with a recent history of a different tumor type were excluded). [0040] Access to the peripheral circulations included a combination of angiocatheters by Arrow (Teleflex Incorporated, USA), or Becton, Dickinson and Company (Franklin Lakes, NJ) placed for intravascular monitoring and infusion, and butterfly needles manufactured by Becton, Dickinson and Company (Franklin Lakes, NJ). Prior to any incision, blood specimens were collected sequentially by first taking a 5cc waste followed by the research specimen. Great care was taken to withdraw blood at approximately the same rate in both circulations (using a lOcc syringe) to avoid a difference in trauma associated with collection.

[0041] Spiked controls were created using blood from consented patients that did not have a diagnosed malignancy who were undergoing procedures that provided arterial and venous access. Human primary lung cancer lines (CRL-5911 and NCI-H520) were cultured and propagated per vendor's recommendations. Cell counts were estimated using micrometer and the cells were reconstituted in PBS. A total of 0.5cc of the cell line concentrate was injected into the arterial and venous blood specimen prior to shipping for central processing.

[0042] Immunomagnetic precipitation (CellSearch^®): 7.5 mL of peripheral blood were collected into Cellsave vials (Cellsave, Immunicon, PA). CTCs were quantified using the CellSearch^® system (Veridex, Raritan NJ) as previously described [1] in the Yale clinical laboratories within 72 hours of collection. Briefly, whole blood was treated with a ferrofluid containing antibodies to the tumor epithelial marker, EpCAM. Magnetic immunoprecipitation was used (based on the iron molecule on EpCAM antibody) to enrich the population of cells containing tumor cells. Antibodies to CK-8, 18, 19 were used to further stain tumor cells while anti-CD45 antibody identifies lymphocytes. The stained cells were then analyzed using a multi-channel fluorescence microscope (Cell-Spotter). In order to be considered a CTC, a nucleus must be present (diamidino-2-phenylindole, DAPI staining), and stained for cytokeratins but not for CD45. Results for this assay were reported as cell number per 7.5 ml. A board certified cytopathologist made the final determination of the CTC.

[0043] Scanning microscopy (Epic sciences method): 8 mL of peripheral blood was collected in a Cell-free DNA blood collection tube (Streck, Omaha, NE). The specimens were stored at room temperature for 4 to 8 hours before being shipped overnight to Epic Sciences central laboratory (San Diego) for processing within 24 hours of collection as previously described in a lung cancer population [18]. In brief, a white blood cell count was taken from each specimen using a hemocytometer and used to titrate cellular concentration to the range in which the assay has been optimized (3 million cells per slide). Red blood cells were lysed using ammonium chloride and the nucleated cells were distributed in a monolayer onto custom glass slides (Epic Sciences). After paraformaldehyde fixation and methanol permeabilization, cells were incubated with anti-Cytokeratin cocktail (recognizing 1,4,5,6,7, 8,10,13,18,19) and anti-CD45 antibodies followed by Alexa 555-conjugated secondary antibody and DAPI as a nuclear stain.

[0044] Each slide was imaged (custom high speed scanning microscope, Epic Sciences at 10X) and "candidate" CTCs were identified as being Cytokeratin positive (CK+), CD45 negative (CD45-) with an intact nucleus using proprietary computer algorithms (Epic Sciences). Each CTC candidate was subsequently evaluated by direct microscopic review of captured images and based on cell morphology and immunophenotype was either confirmed or rejected as being a CTC by two independent reviewers. Each CTC cluster was counted as single CTC, however the number of clusters (>2 cells) were tracked for each patient.

[0045] CTC counts were reported per milliliter of blood specimen (CTCs/ml), and reflect titrations made to optimize cellular concentration as described above.

Statistics

[0046] Paired observations (arterial and venous) for each patient were evaluated using a paired t test with p values of < 0.05 considered to be significant.

Results

[0047] Immunomagnetic precipitation (CellSearch^®): the initial phase of this study was carried out using CellSearch^® assay in a patient population that included metastatic cancer (12 patients) originating in colon or rectum, or pancreas, prostate, appendix. The study transitioned to primary lung cancer (9 patients) based on the rationale outlined above. Of the 21 patients evaluated with the CellSearch® Assay, CTCs were only detected in 10 patients, which are shown in Table 3. Γη the single patient with a difference beyond what could be expected in normal interassay variability, more cells were identified in the venous sample (69) than arterial (0). Table 3. Identification of CTCs by Immunomagnetic Precipitation

Single patient with a difference that would be greater than expected inter-assay variability

[0048] Scanning microscopy (Epic sciences method): In order to increase the sensitivity of the study, Epic sciences method (an assay with a greater range of detection in solid tumors) that is based on scanning microscopy was used. A total of 35 patients with primary lung cancer were studied, representing a range of histologies and stages as described in Table 4. A significant difference was seen in the prevalence of CTCs between arterial and venous specimens (Figure 2) with arterial specimens having fewer cells (p = 0.002).

Table 4. Scanning Microscopy population demographics

^represents best stage determination by available information with pathologic being better than clinical

** patients presumed to be at a resectable stage, but found intraoperatively to have more advanced disease. [0049] Clusters of two or more CTCs were also less common in the arterial blood p = 0.001. In addition, larger clusters of 10 or more cells were seen in 12 venous samples, but only 3 arterial samples.

[0050] Attempts to exclude artifacts: Several steps were taken to address potential sources of artifacts. In order to exclude the impact of access size (as venous access often uses larger caliber than arterial access), 17 patients were examined with either the identical access diameter (n = 14) or with a venous access that was smaller than arterial (N = 3). CTCs continued to be less common in arterial blood specimens (p = 0.004). In order to evaluate the impact of the access type and manufacturer, 11 patients were examined using identical catheters or needles in the arterial and venous circulations, and the differences remained (p = 0.01).

[0051] In order to evaluate if the arterial conditions affected CTCs ex vivo viability, or if the arterial conditions were affecting the Epic sciences method, arterial and venous blood was taken from patients without a cancer history and spiked with cultured primary lung cancer cell lines. The arterial conditions did not appear to alter the counts of tumor cells (Table 5). In order to determine whether "arterialized" CTCs themselves were less stable for transportation, a set of matched specimens were processed immediately and shipped overnight to the central lab which did not significantly alter the results (Table 6).

Table 5. Spiked Controls

Cell Line Injected cell # Arterial Venous (cells/cc)

(cells/cc)

CRL-5911 3 - 4,000 cells 45 37

Adenocarcinoma

NCI - H520 Squamous 3 - 4,000 cells 455 361

Cell Carcinoma

NCI - H520 Squamous 3 - 4,000 cells 245 235

Cell Carcinoma Table 6. Freshly prepared and shipped cells

[0052] Left heart catheterized patients: In order to evaluate the impact of pressure and flow kinetics on the arterial CTC counts, two patients that were undergoing left heart catheterization were evaluated, as these were known to fluctuate along the course of arterial blood transit. Although this is highly select patient population, there did appear to be a trend towards more cells in the blood specimens drawn closer to the heart (Figure 3A-B). The numbers in the figure reflect the number of cells obtained from the different sources in the body (aorta, radial artery, descending aorta, femoral artery, and venous). Unfortunately a venous specimen was not possible from the second patient.

[0053] There are several possible explanations for the observed differences. The first possible explanation would be that the arterial population is not being accurately evaluated in this study (arterial cells are not accessible, or that cells are being destroyed during collection or processing). Multiple steps have been taken to exclude any differences that may relate to collection, most notably in the 11 patients whose arterial and venous specimens were obtained using identical access (size and manufacturer), as well as excluding differences relating to transportation, or processing. Even among the heart catheterized patients, the arterial cell counts were at most equal to what was expected on the venous side (Table 7). Therefore no hypothesized drop off between arterial and venous blood was observed to implicate the capillary bed as restricting CTC flow. Table 7. Patients with identical access for arterial and venous blood draw

*patient numbers are unique to each table and do not carry over between figures

BD - Braun Becton, Dickinson and Company (Franklin Lakes, NJ)

Arro - Arrow (Teleflex Incorporated, USA)

Bfly- butterfly needle manufactured by Becton, Dickinson and Company (Franklin Lakes, NJ)

[0054] The second possibility would be a true difference in the prevalence of circulating tumor cells with fewer cells existing in the arterial circulation. CTCs are known to exist immediately upstream of the systemic arterial circulation in the pulmonary veins of lung cancer patients [19-23]. Therefore, a reduction in cells in the arterial circulation would imply cell loss via destruction or spontaneous death. This could reflect a fragility of CTCs rendering them more vulnerable to conditions on the arterial side than in the pulmonary veins immediately preceding the heart. However a loss of arterial cells would imply a downstream expansion of the CTC pool in the venous circulation. Potential sources of this expansion could be an alternate circulation, cell division among venous cells or finally shedding of tumors cells into the venous circulation by a clinically occult reservoir.

[0055] In this example, potential sources of artifact relating to the way in which blood was retrieved, variations in conditions (pH, oxygen level, etc.) between arterial and venous blood, and potential differences in the stability of the cells themselves were examined. Our study suggested that the finding of fewer CTCs in the arterial circulation could reflect a difference in availability of the cells in the arterial circulation (cells are traveling via arteries, but not in part of the vessel or in a form that is able to be extracted by the access needle or catheter). If the venous circulation were truly to have greater numbers, this would implicate either a different drainage pattern for tumors, collateral pathways that do not include the arterial circulation, or¾ source of CTCs between the arteries and veins (such as tumor emboli in the capillaries that shed cells without ever invading or forming clinical metastases. We have "arterialized" CTCs. The finding of lower counts of CTCs in the arterial circulation should be taken into consideration as the use of these assays increase, particularly in a surgical population, where arterial blood is often the easiest to access.

EXAMPLE 4

[0056] In this example, quantities of CTCs in the arterial and venous circulations of advanced non-small cell lung cancer (NSCLC) patients are examined using Epic Sciences technology. In addition, differences in the morphology, size, cellular association (clusters), and protein expression in tumor cells derived from the arterial and venous circulations of NSCLC patients are also characterized using the Epic Sciences technology.

[0057] 1 tube of peripheral arterial and 1 tube of peripheral venous blood are obtained from each eligible patient identified and consented. The Epic blood sample is processed in accordance with provided protocol and collection materials, and transported in a blinded fashion marked only with a Yale research identifier. The Epic blood sample is devoid of information that contains protected health information, or the source of the blood (arterial or venous). This information contains a white blood cell (WBC) count that is associated with the patient.

EXAMPLE 5

[0058] To determine whether the differences in CTCs obtained in venous blood and arterial blood were affected by the arterial pressures. Patients undergoing heart catheterizations are examined in order to determine by using a larger catheter, and by obtaining the blood more centrally.

[0059] All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0060] As will be appreciated by one of skill in the art, while the present specification may simply use one of the terms "comprise," "consists," or "consists essentially of," this is simply a shorthand way of describing all three possibilities, unless otherwise specified or unless the term is used in a claim (in which case the terms will have their normally accepted meanings under claim interpretation). Thus, as the terms are used above, they designate all three possibilities, unless explicitly noted otherwise.

[0061] References:

1. Allard, W.J., et al., Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases.

2004. p. 6897-6904.

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Claims

WHAT IS CLAIMED IS:

1. A method for analyzing rare cells in blood, comprising: providing a sample of blood from a subject in a container with at least one preservative, wherein the sample is obtained from the subject using a needle or catheter that is 18 gauge or larger; identifying at least one rare cell in the sample; and analyzing the rare cell at least about 6 hours after obtaining the sample from the subject.

2. The method of Claim 1, wherein the needle or catheter is 16 gauge or larger.

3. The method of Claim 1, wherein the needle or catheter is 14 gauge or larger.

4. The method of Claim 1, wherein the sample is arterial blood.

5. The method of Claim 1, wherein the sample is venous blood.

6. The method of Claim 1, wherein said rare cells are selected from the group consisting of circulating tumor cells (CTCs), circulating endothelia cells (CECs), fetal cells, stem cells, and any combination thereof.

7. The method of Claim 1, wherein analyzing said rare cell comprises identifying circulating tumor cells (CTCs).

8. The method of Claim 1, wherein the rare cell is analyzed about 6 to about 8 hours after the sample is obtained from the subject.

9. The method of Claim 1, wherein identifying the rare cell comprises an immunochemical analysis.

10. The method of Claim 1, wherein analyzing the rare cell comprises an analysis selected from the group consisting of genomics analysis, epigenomics analysis, transcriptomics analysis, proteomics analysis, and any combination thereof.

11. The method of Claim 1, further comprising enriching rare cells in the sample.

12. The method of Claim 1, further comprising contacting the sample with an agent capable of maintaining the structural integrity of the rare cell in the sample.

13. The method of Claim 12, wherein the structural integrity of the rare cells is determined by a procedure selected from the group consisting of immunocytochemical procedures, RT-PCR procedures, PCR procedures, FISH procedures, flowcytometry procedures, image cytometry procedures, and any combination thereof.

14. A kit for collecting a blood sample that includes rare cells in blood, comprising: a needle or catheter that is 18 gauge or larger; and a container with at least one agent capable of maintaining the structural integrity of the rare cell in the sample.

15. The kit of Claim 14, wherein the needle or catheter is 14 gauge or larger.