• prostate cancer;
  • blood;
  • flow cytometry;
  • fluorescence microscopy;
  • image analysis


  1. Top of page
  2. Abstract
  6. Acknowledgements


The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status.


In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5-ml blood samples collected from patients with prostate cancer in evacuated blood-draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6-diamidino-2-phenylindole.


In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6-diamidino-2-phenylindole and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples.


The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients. © 2004 Wiley-Liss, Inc.

Circulating tumor cells (CTCs) can be detected in the peripheral blood of patients with cancer (1–8). Proof that these cells originate from the tumor was demonstrated by identification of the same chromosomal aberrancies in the primary tumor and CTCs (9). More importantly the presence of CTCs has been associated with a shortened survival rate (10–17). The actual number of CTCs reported in the blood of cancer patients, however, varies greatly between studies. Fragility of the CTCs, different sample preparation and analysis techniques, and variability caused by the laborious manual process used to classify CTCs are the most likely source of these discrepancies. To characterize the composition of CTCs, the integrity of CTCs needs to be maintained after the blood is drawn and during the sample processing (18, 19). This was achieved by blood collection in CellSave Preservative Tubes. To minimize variability in the sample preparation process, a semi-automated sample preparation system was used to immunomagnetically enriched CTCs from 7.5-ml blood samples targeting the epithelial cell adhesion molecule (EpCAM) (20). In this study, we compared enumeration of immunomagnetically enriched CTCs from patients with metastatic prostate cancer by using flow cytometry and fluorescence microscopy. We found that a substantial proportion of CTCs detected were undergoing apoptosis, as demonstrated by the presence of the M30 epitope present only on caspase-cleaved cytokeratin 18, or were identified as tumor cell fragments.


  1. Top of page
  2. Abstract
  6. Acknowledgements


Ten patients with metastatic prostate cancer were included in this study. Patient demographics are presented in Table 1. Patients and healthy male volunteers signed an informed consent form under an approved research study. Blood was drawn into 10-ml CellSave Preservative Tubes that contained ethylene-diaminetetra-acetic acid as a anticoagulant and a cellular preservative (Immunicon Corp., Huntington Valley, PA). Samples were kept at room temperature and processed within 72 h.

Table 1. Enumeration of CTC by Fluorescent Microscopy and Flow Cytometry in Blood Samples of 10 Patients with Prostate Cancer and 10 Healthy Males
Patient no.Patient clinical informationAll CTCsaIntact CTCsaDamaged CTCsaCTC fragmentsa
AgeTherapybPSA (ng/ml)StatuscCSFCCSFC DAPIpos M30negFC DAPIpos M30posCSFC DAPIdim M30posCSFC DAPIdim M30neg
  • a

    Data presented as number of events detected (% subpopulation of events). CS, CellSpotter fluorescent microscopis analysis; FC, flow cytometric analysis, PSA, prostate-specific antigen.

  • b

    Ca, Casodex; Cy, Cytoxan; Ds, Diethylstilbestrol; Lu, Lupron; Mi, Mitoxantrone; Pr, Prednisone; RT, radiation therapy; Th, Thalidomide; To, Topotocean; Tx, Taxotere; Zo, Zometa.

  • c

    P, progressing; S, stable; R, Responding.

176Lu60P22921 (4)1 (1)8 (9)7 (32)51 (55)14 (64)32 (35)
249To0R1001254 (4)0 (0)12 (9)12 (12)102 (82)84 (84)11 (9)
362Cy/Pr6S11220010 (9)15 (7)11 (6)16 (14)37 (18)86 (77)137 (69)
463Lu58P19226616 (8)27 (10)39 (15)2 (1)91 (34)174 (91)109 (41)
571Lu/Ca150P54434716 (3)10 (3)4 (1)38 (7)34 (10)490 (90)299 (86)
666Tx2,000P37622316 (4)2 (1)26 (12)40 (11)165 (74)320 (85)30 (13)
775Mi/Th198P293622 (76)11 (31)5 (14)5 (17)9 (25)2 (7)11 (31)
874Tx/Zo113S34234732 (9)3 (1)31 (9)56 (17)160 (46)254 (74)153 (44)
986Di250P45848540 (9)41 (8)14 (3)38 (8)82 (17)380 (83)348 (72)
1090RT433S498658240 (48)224 (34)19 (3)20 (4)159 (24)238 (48)256 (39)
Mean71     (17)(10)(7)(12)(39)(71)(41)
     y = 0.80x + 64y = 0.96x + 12y = 1.56x + 52y = 0.58x + 20
     R2 = 0.71R2 = 0.95R2 = 0.25R2 = 0.57
Normal donors             

Sample Preparation

A sample preparation system designed to process 7.5 ml of blood and described in detail elsewhere (20) was used to select and concentrate epithelial cells present in the blood sample. In brief, magnetic nanoparticles labeled with monoclonal antibodies identifying EpCAM were used to enrich epithelial cells. The magnetically captured cells were resuspended in a volume of 200 μl and fluorescently labeled. To identify epithelial cells, phycoerythrin-labeled monoclonal antibodies recognizing keratins 8 and 18 and another monoclonal antibody that recognizes keratin 19 were used (CK-PE). Allophycocyanin-labeled monoclonal antibodies that recognize CD45 (CD45-APC) were used to discriminate epithelial cells from leukocytes. The double-stranded DNA-specific dye 4,6-diamidino-2-phenylindole (DAPI) was used to visualize and characterize the nucleus. After incubation, the captured cells were resuspended and transferred into an analysis cartridge contained within a magnetic yoke assembly that holds the cartridge between two magnets (MagNest™, Immunicon Corp.). The shape of the magnets is designed such that magnetically labeled cells present in the cartridge move vertically to the viewing surface. The viewing surface is positioned approximately 3 mm below the surface of the magnets. At this position, the magnetic gradient is still vertical and prevents horizontal movement of the cells, resulting in a homogenous distribution of the cells over the viewing surface (20).

After analysis of the sample by microscopy, the cell suspension was removed from the cartridge and placed in a 12- × 75-mm tube. Residual cells were removed from the cartridge by repeated washing of the cartridge with 1 ml of phosphate buffered saline containing 0.5% bovine serum albumin. The tube was placed in a QMS13 to magnetically wash the sample for 10 min (Immunicon Corp.). The cells were resuspended in 200 μl of phosphate buffered saline plus 0.5% bovine serum albumin and incubated for 15 min with 20 μl (1 μg) of the fluorescein isothiocyanate–conjugated M30 monoclonal antibody (M30 FITC; Roche Applied Science, Manheim, Germany). The M30 antibody was obtained from experiments designed to create a new CK18 antibody that appeared to stain apoptotic epithelial cells. Further analysis showed that the antibody reacted with a neo-epitope expressed only after caspase cleavage of CK18 during early apoptosis (21). After incubation, the cells were washed in a QMS13 and resuspended in 300 μl of CellFix (Immunicon Corp.) and analyzed by flow cytometry.

Sample Analysis by Fluorescence Microscopy

The CellSpotter™ Analyzer, a semi-automated fluorescence microscope system, was used for analysis (Immunicon Corp.). The system consists of a Nikon E-400 microscope with a mercury arc lamp, a 10× objective (Warming distance 4 mm, numerical aperture 0.45), a high-resolution X, Y, Z stage, and a four filter cube changer controlled with a Ludl MAC2002 controller. Excitation, dichroic, and emission filters in the four cubes are 365 nm/400 nm/400 nm, 480 nm/495 nm/510 nm, 546 nm/560 nm/580 nm, and APC 620 nm/660 nm/700 nm. Cells are distributed over a surface area of 80.2 mm2, and images are acquired with a Hamamatsu 12-bit, 1,280- × 1,024-pixel digital camera connected to a National Instruments PCI-1424 digital frame grabber. The entire surface of the cartridge is scanned for each of the four filters, resulting in 560 images. The CellSpotter acquisition program automatically determines the region over which the images are to be acquired, the number of images to acquire, the position of each image, and the microscope focus to use at each position. All images from a sample are logged into a directory that is unique to the specific sample identification. The data analysis and presentation program was written using IBM's DB-2 database through an HTML interface. An algorithm is applied on all of the images acquired from a sample to search for locations that stain for DAPI and CK-PE. If the staining area is consistent with that of a potential tumor cell (DAPI+, CK-PE+), the software stores the location of these areas in a DB-2–based database. The software displays thumbnails of each of the boxes, and the user can place a check beside them to confirm that the images represented in the row are consistent with either tumor cells or leukocytes (CD45+).

Sample Analysis by Flow Cytometry

Samples were analyzed on an LSR flow cytometer equipped with a 488-nm argon-ion laser, 633-nm helium-neon laser, and a 325-nm helium-cadmium laser (BDIS, San Jose, CA). Data acquisition was performed with CellQuest (BDIS) and was stopped after the entire contents of each tube were collected. Multiparameter data analysis was performed on listmode data (CellQuestPro, BDIS). Analysis criteria included positive staining with the CK-PE, no staining with CD45-APC, and variable staining with DAPI and FITC-labeled M30 antibody.

Cell Culture and Cell Spiking

Cells of the prostate cancer cell line LNCaP were cultured in flasks containing 10 ml of RPMI-1640 supplemented with 10% fetal calf serum. Twenty-four hours after passage, 40 nM of paclitaxel (Cytoskeleton Inc., Denver, CO) was added to the culture medium and allowed to incubate at 37°C for an additional 72 h. A flask of untreated cells was run concurrently. After incubation, both flasks were treated with trypsin and combined with cells that had detached from the flask and were washed. The cell viability was assessed using trypan blue exclusion followed by a serial dilution. To determine the cell number, a 100-μl aliquot of the fresh or paclitaxel treated LNCaP cells was permeabilized and stained with CK-PE. Standardized fluorescent beads (Flow Set Fluorospheres, Beckman Coulter, Miami, FL) were added to the sample and analyzed by flow cytometry. The absolute number of cytokeratin-positive events in the sample was determined by using the absolute bead count. CellSave tubes were used to collect whole blood from healthy volunteers, and 100 μl of LNCaP cells was spiked into 7.5 ml of the sample. The samples were incubated for 24 h and then isolated by immunomagnetic separation.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Characterization of CTC by Fluorescent Microscopy

Figure 1 shows an example of CellSpotter analysis of a blood sample from a patient with metastatic prostate cancer. Regions that potentially contain CTCs are displayed in rows of thumbnails. The scale bar in the left lower corner of the figure indicates the size of the thumbnails. From right to left, these thumbnails represent nuclear (DAPI), cytoplasmic cytokeratin (CK-PE), control (no reagent), and surface CD45 (CD45-APC) staining. The composite images display a false-color overlay of the magenta nuclear (DAPI) and green cytoplasmic (CK-PE) staining. The check box beside the composite image allows the user to confirm that the images represented in the row are consistent with a tumor cell, and the check box beside the CD45-APC image is used to identify a leukocyte or tumor cell that stained nonspecifically. In this patient sample, 2,761 rows of thumbnails were detected by the software that demonstrated staining consistent with tumor cells. Of the 2,761 rows, 12 are shown in Figure 1 and labeled 1632–1638 and 1869–1873. Rows numbered 1636, 1638, and 1873 are checked off and display features of intact CTCs defined as a size larger than 4 μm, the presence of a nucleus surrounded by cytoplasmic cytokeratin staining, and absence of control and CD45 staining. Note the difference in appearance of the tumor cells; the cell in row 1638 is large and the one in row 1636 is significantly smaller. The immunophenotype of the event in row 1869 is consistent with a tumor cell, but the morphology shows a large nucleus with speckled cytoplasm due to retraction of cytoskeletal proteins. The thumbnail in row 1634 shows a damaged cell that appears to extrude its nucleus. The thumbnail in row 1632 shows a cell that stains with cytokeratin and CD45 and is either a tumor cell nonspecifically binding to CD45 or a leukocyte nonspecifically staining with cytokeratin. In this instance, the morphology of the cell closely resembles that of a lymphocyte. The thumbnails shown in rows 1633, 1635, 1637, 1870, and 1872 shows cytokeratin staining objects that are larger than 4 μm but have no resemblance to cells. The cytokeratin-positive objects in thumbnails 1637 and 1872 are in close proximity of a leukocyte. Based on the observation of images of CTC candidates in several patient samples, CTCs were classified into three categories: intact CTCs, damaged CTCs, and CTC fragments that did not stain with CD45 or appear in the control channel.

thumbnail image

Figure 1. Example of a fluorescent microscopy CTC analysis by CellSpotter from a 7.5-ml blood sample from a patient with metastatic prostate cancer. The columns of thumbnails correspond to the images from corresponding fields of the nucleus (DAPI), cytokeratin (CK-PE), control, leukocyte (CD45-APC), and false-color overlay image of cytokeratin (green) and the nucleus (magenta). The rows of numbered thumbnails present the different locations in the analysis cartridge that contain CTC candidates. The center box of the “gun sight” in the lower right corner is 4 μm and is used to size individual events during review of the images to ensure a minimal cell size of 4 μm.

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Characterization of CTC by Flow Cytometry

Figure 2 shows an example of the flow cytometric analysis of a blood sample from a patient with metastatic prostate cancer that was previously analyzed by fluorescent microscopy. Figure 2A shows the CD45-APC and CK-PE staining of all events in the sample, and Figure 2B shows the DAPI and CK-PE staining. The red-colored events that stain with CD45-APC and have a normal DNA content as demonstrated by their presence in the lower right-hand quadrant of Figure 2B are intact leukocytes. The green-colored events that are positive for cytokeratin and are present in the upper right-hand quadrant of Figure 2B are CTCs with normal DNA content, and the black and magenta events that are positive for cytokeratin are CTCs with insufficient DNA content. All other events are depicted in gray. Figures 2C and 2D are from the same experiments, but only the cytokeratin-positive events are shown. The CK-PE–positive and CD45-APC–negative events were subdivided based on staining with M30 FITC and DAPI (Fig. 2D). Events that were positive for DAPI and negative for M30 are shown in green. Events that were positive for DAPI and M30 are shown in orange. Events that were negative for DAPI and positive for M30 are shown in black. Events that were negative for DAPI and M30 are shown in magenta. The arrow in Figure 2D indicates the apoptotic pathway of CTCs.

thumbnail image

Figure 2. Flow cytometric CTC analysis of a sample previously analyzed by microscopy. A and B shows all events; C and D show only the cytokeratin-positive events. CD45+ events in A and B are colored red and debris is shown in gray. Based on staining with M30 FITC and DAPI in D, events that were positive for DAPI and negative for M30 are shown in green. Events that were positive for DAPI and M30 are shown in orange. Events that were negative for DAPI and positive for M30 are shown in black. Events that were negative for DAPI and M30 are shown in magenta. Arrow in D indicates the apoptotic pathway of CTCs.

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Evaluation of CTC in Prostate Cancer Patients by Fluorescent Microscopy and Flow Cytometry

Figure 3 shows the analysis of CTCs by fluorescent microscopy and flow cytometry from blood samples of patients 10, 6, and 5. Individual thumbnails from the CTC images are shown as a composite and divided into three morphologically distinct categories, separated by the red bars from left to right: intact CTCs, damaged CTCs, and CTC fragments. The flow cytometry plot of DAPI versus M30 FITC staining of the cytokeratin-positive events of the same sample is shown to the right of the collection of images. The number and relative frequency of the DAPIpos,M30neg, DAPIpos,M30pos, DAPIdim,M30pos, and DAPIdim,M30neg events are provided in the figure.

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Figure 3. Fluorescent microscopy and flow cytometric CTC analysis in three patients with prostate cancer. A: For patient 10, 53 of 240 intact CTCs, 7 of 20 damaged CTCs, and 19 of 238 CTC fragments are shown. Green represents cytokeratin and magenta the nucleus. B: The corresponding flow cytometric analysis of only the cytokeratin-positive events is presented. C: For patient 6, 2 of 16 intact CTCs, 33 of 40 damaged CTCs, and 6 of 320 CTC fragments are shown. D: Corresponding flow cytometric analysis. E: For patient 5, 2 of 16 intact CTCs, 15 of 38 damaged CTCs, and 49 of 490 CTC fragments are shown. F: Corresponding flow cytometric analysis.

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In the blood of patient 10 shown in Figure 3A, most CTCs are intact and defined as objects larger than 4 μm, with a relatively smooth cytoplasmic membrane, cytoskeletal proteins throughout the cytoplasm, and an intact nucleus encompassed within the cytoplasm. This population most likely correlates with the DAPIpos,M30neg and DAPIpos,M30pos regions on the corresponding flow plot shown in Figure 3B. Fewer CTCs were identified as damaged or cell fragments.

In the blood of patient 6, shown in Figure 3C, most CTCs found by microscopy consisted of damaged CTCs defined as objects larger than 4 μm, with speckled cytokeratin staining or ragged cytoplasmic membrane and a nucleus associated with the cytokeratin. The speckled cytokeratin staining is most likely the result of cytoskeletal retraction and degradation associated with the apoptotic cascade. Consistent with this is the finding by flow cytometry that most events (74%) are DAPIdim and M30pos, indicating that these cells are in the process of apoptosis (Fig. 3D).

In the blood of patient 5, shown in Figure 3E, most CTCs found by microscopy were cell fragments defined as round cytokeratin positive objects of at least 4 μm with or without association of nuclear material that had no morphologic resemblance to a cell. The morphology of the objects is consistent with cell fragments likely representing apoptotic bodies at the end of the apoptosis process. This is consistent with this finding that most events by flow cytometry (86%) are DAPIdim and M30neg (Fig. 3F).

Table 1 summarizes the findings obtained by microscopy and flow cytometry of each of the 10 patients. In addition, the analysis of blood samples from healthy men is shown in the table. In these samples, 0 events were classified as intact CTCs by microscopy and no more than two were detected by flow cytometry. In some of the healthy donors, few events were found that were classified as damaged or CTC fragments. The correlation between both analytic methods to detect CTC in patient samples was R2 = 0.71, indicating that, although the same samples were analyzed, the same numbers of CTCs were not detected. For detection of intact CTCs, the correlation increased to R2 = 0.95. The number of intact CTCs detected by flow cytometry was significantly larger (P = 0.0247), but this may be attributed to the inclusion of CTCs expressing M30. Only a small percentage of all cytokeratin positive events was classified as intact CTCs by microscopy (17%) and flow cytometry (10%, M30neg and 7%, M30pos) with the majority representing CTC fragments. The variation in the relative proportion of intact CTC between patients was large, ranging from 3% to 76% by microscopy. No or poor correlation was found in detection of damaged CTCs and CTC fragments between flow cytometry and microscopy.

In Vitro Induction of Apoptosis in LNCaP cells

To investigate the effect of apoptosis induced by cytotoxic agents on flow cytometric and fluorescent microscopic analyses of CTCs, cells from the prostate cell line LNCaP were cultured in the presence or absence of 40 nM paclitaxel for 72 h. Viabilities assessed by trypan blue exclusion after incubation of untreated LNCaP cells were 91% and 35% for the paclitaxel-treated cells. The treated and untreated LNCaP cells were spiked into blood of healthy donors. After immunomagnetic selection and fluorescent labeling, the cells were analyzed by fluorescent microscopy. More than 90% of the LNCaP cells that were not treated with paclitaxel were classified as intact tumor cells (Fig. 4A) whereas most paclitaxel-treated cells showed features consistent with apoptosis (Fig. 4C). After microscopic analysis, the cells were retrieved from the cartridge, stained with M30 FITC, and analyzed by flow cytometry. The correlative display of the DAPI and M30 FITC stainings by flow cytometry of the untreated cells that expressed cytokeratin is shown in Figure 4B and that of the treated cells is depicted in Figure 4D. Most cells by flow cytometry have intact DNA content (75%), and 24% of these cells express M30, indicating that they have started the process of apoptosis. In contrast to the untreated LNCaP cells, the paclitaxel-treated cells displayed speckled cytokeratin staining and irregular morphology. By flow cytometry, the predominant population shows decreased DAPI staining (87%), and 26% of these cells express M30. The apoptotic pattern of CTCs in the patient samples closely resembles that of the paclitaxel-treated LNCaP cells.

thumbnail image

Figure 4. Fluorescent microscopy and flow cytometric CTC analysis of LNCaP cells spiked in blood of normal donors. A: Images of untreated LNCaP cells. B: Corresponding flow cytometric analysis of only the cytokeratin-positive events. C: Images of paclitaxel-treated LNCaP cells. D: Corresponding flow cytometric analysis.

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  1. Top of page
  2. Abstract
  6. Acknowledgements

The presence of tumor cells in blood from patients with metastatic carcinoma has been associated with shortened survival rate (10–17), and their presence in bone marrow from patients diagnosed with cancer has been associated with poorer prognosis (22–25). These studies were hampered by methods lacking reproducibility, sensitivity, and specificity. In addition, the criteria used to identify tumor cells varied, thereby making a comparison of outcomes difficult at best. These problems have been recognized, and attempts are being made to standardize assay methods and the analytical criteria used to define a tumor cell (26). In this study, the limitations associated with operator variability were overcome by the use of a semi-automated sample preparation system (20), and issues concerning the identification of cells were minimized by analyzing samples by flow cytometry and fluorescence microscopy. Blood samples were immunomagnetically enriched for cells that express EpCAM, and CTCs were 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 (DNA+).

Another major obstacle to detect CTCs reliably is their fragility as evidenced by a significant loss of CTCs within 24 h of blood draw (18, 19). The question arises as to whether the reported apoptosis in a substantial portion of CTCs (27, 28) is due to degradation before or after the blood draw. To circumvent this, blood in this study was collected in evacuated blood-draw tubes that contained an anticoagulant and a preservative, thereby maintaining the integrity of the cells.

With the use of the preservative, only 17% (range 3% to 76%) of the CK+, CD45, and DNA+ objects detected by fluorescence microscopy in blood from 10 patients with metastatic prostate cancer resembled intact cells. The other objects appeared to be fragments or damaged cells with a cytokeratin staining pattern that suggested a collapse of the cytoskeleton and can be attributed to caspase activation resulting in cleavage of keratin 18 during epithelial cell apoptosis (29, 30). This was indeed the case, as evidenced by the staining with the M30 cytodeath antibody of these cells. M30 recognizes an epitope of cytokeratin 18 that is exposed only after caspase cleavage in early apoptosis (21). The cytokeratin-positive, M30-positive events contained normal and low DNA content. This is in agreement with the observation of nuclear material containing M30-positive fragments in the spleens of patients with metastasized cancer (31). Similar patterns of apoptosis were observed after treatment of cells of the prostate cell line LNCaP with paclitaxel, an agent that induces apoptosis.

The detection of the presence or absence of proteins and genes targeted by antineoplastic agents in CTCs may provide the opportunity to tailor treatment (32, 33). Persistence of CTCs 3 to 4 weeks after initiation of therapy strongly suggests that these patients are on futile therapy (34). Many cytotoxic and target-directed therapeutics function by inducing apoptosis in carcinoma cells. Determination of the proportion of CTCs undergoing apoptosis after administration of antineoplastic agents could further improve the ability to measure effectiveness of therapy and may lead to the acceleration of the development and utilization of novel therapeutics.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Special thanks to Gina DiTonno, Maxine Giannotti, and Jason Rowand for assistance in patient sample preparation.


  1. Top of page
  2. Abstract
  6. Acknowledgements
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