A nanowell platform to identify, sort and expand high antibody-producing cells.

Increased use of therapeutic monoclonal antibodies and the relatively high manufacturing costs fuel the need for more efficient production methods. Here we introduce a novel, fast, robust, and safe isolation platform for screening and isolating antibody-producing cell lines using a nanowell chip and an innovative single-cell isolation method. An anti-Her2 antibody producing CHO cell pool was used as a model. The platform; (1) Assures the single-cell origin of the production clone, (2) Detects the antibody production of individual cells and (3) Isolates and expands the individual cells based on their antibody production. Using the nanowell platform we demonstrated an 1.8-4.5 increase in anti-Her2 production by CHO cells that were screened and isolated with the nanowell platform compared to CHO cells that were not screened. This increase was also shown in Fed-Batch cultures where selected high production clones showed titers of 19-100 mg/L on harvest day, while the low producer cells did not show any detectable anti-Her2 IgG production. The screening of thousands of single cells is performed under sterile conditions and the individual cells were cultured in buffers and reagents without animal components. The time required from seeding a single cell and measuring the antibody production to fully expanded clones with increased Her-2 production was 4-6 weeks.

Isolation of Single Circulating Tumor Cells Using VyCAP Puncher System.

Tumor heterogeneity has a major role in the development of tumor evasion and resistance to treatments. To study and understand the intrinsic heterogeneity of cancer cells, the use of single-cell isolation technology has had a major boost in recent years, gaining ground to bulk analysis in the study of solid tumors. In the liquid biopsy field, the use of technologies for single-cell analysis has represented a major advance in the study of the heterogeneity of circulating tumor cells (CTCs), providing relevant information about therapy-resistant CTCs. However, single-cell analysis of CTCs is still challenging due to the weakness and scarcity of these cells. In this chapter, we describe a protocol for CTCs isolation at a single-cell level using the VyCAP Puncher system.

Measurement of the Drug Sensitivity of Single Prostate Cancer Cells.

The treatment of cancer faces a serious challenge as cancer cells within patients are heterogeneous and frequently resistant to therapeutic drugs. Here, we introduce a technology enabling the assessment of single cancer cells exposed to different drugs. PCa cells were individually sorted in self-seeding microwells, cultured for 24 h, and then exposed to several drugs to induce (R1881) or inhibit (Enzalutamide/Abiraterone) the secretion of a protein (PSA). Cell viability and PSA secretion of each individual prostate cell were monitored over a 3-day period. The PSA protein secreted by each cell was captured on a PVDF membrane through a pore in the bottom of each well. The basal PSA secretion was found to be 6.1 ± 4.5 and 3.7 ± 1.9 pg/cell/day for LNCaP and VCaP, respectively. After exposure to R1881, the PSA secretion increased by ~90% on average and was not altered for ~10% of the cells. PSA production decreased in the majority of cells after exposure to enzalutamide and abiraterone.

Microfluidic device for DNA amplification of single cancer cells isolated from whole blood by self-seeding microwells.

Self-seeding microwell chips can sort single cells into 6400 wells based on cell size and their identity verified by immunofluorescence staining. Here, we developed a microfluidic device in which these single cells can be placed, lysed and their DNA amplified for further interrogation. Whole blood spiked with MCF7 tumor cells was passed through the microwell chips after leukocyte depletion and 37% of the MCF7 cells were identified by epithelial cell adhesion molecule (EpCAM) staining in the microwells. Identified single cells were punched into the reaction chamber of the microfluidic device and reagents for cell lysis and DNA amplification introduced sequentially by peristaltic pumping of micro-valves. On-chip lysis and amplification was performed in 8 parallel chambers yielding a 10,000 fold amplification of DNA. Accessibility of the sample through the reaction chamber allowed for easy retrieval and interrogation of target-specific genes to characterize the tumor cells.

The detection of EpCAM(+) and EpCAM(-) circulating tumor cells.

EpCAM expressing circulating tumor cells, detected by CellSearch, are predictive of short survival in several cancers and may serve as a liquid biopsy to guide therapy. Here we investigate the presence of EpCAM(+) CTC detected by CellSearch and EpCAM(-) CTC discarded by CellSearch, after EpCAM based enrichment. EpCAM(-) CTC were identified by filtration and fluorescent labelling. This approach was validated using different cell lines spiked into blood and evaluated on blood samples of 27 metastatic lung cancer patients. The majority of spiked EpCAM(+) cells could be detected with CellSearch, whereas most spiked cells with EpCAM(low) or EpCAM(-) expression were detected using filtration. Five or more CTC were detected in 15% of the patient samples, this increased to 41% when adding the CTC detected in the discarded blood. The number of patients with CTC and the number of CTC detected were doubled by the presence of EpCAM(-) CTC. In this pilot study, the presence of EpCAM(+) CTC was associated with poor outcome, whereas the EpCAM(-) CTC were not. This observation will need to be confirmed in larger studies and molecular characterization needs to be conducted to elucidate differences between EpCAM(-) and EpCAM(+) CTC.

Self-seeding microwell chip for the isolation and characterization of single cells.

A self-seeding microwell chip is introduced for the isolation and interrogation of single cells. A cell suspension is transferred to a microwell chip containing 6400 microwells, each microwell with a single 5 µm pore in the bottom. The fluid enters the microwell and drags a cell onto the pore. After a cell has landed onto the pore, it will stop the fluid flow through this microwell. The remaining fluid and cells will be diverted to the next available microwell. This results in a fast and efficient distribution of single cells in individual microwells. After identification by fluorescence microscopy, the cells of interest are isolated from the microwell by punching the bottom together with the cell. The overall single cell recovery of seeding followed by isolation of the single cell, is >70% with a specificity of 100% as confirmed by the genetic make-up of the isolated cells.

Importance of circulating tumor cells in newly diagnosed colorectal cancer.

Presence of circulating tumor cells (CTC) is associated with poor prognosis in patients with metastatic colorectal cancer (CRC). The present study was conducted to determine if the presence of CTC prior to surgery and during follow­up in patients with newly diagnosed non-metastatic CRC can identify patients at risk for disease recurrence. In a prospective single center study 183 patients with newly diagnosed non-disseminated CRC, scheduled for surgery, were enrolled and followed-up for a median of 5.1 years. CTC were enumerated with the CellSearch system in 4 aliquots of 7.5 ml of blood before surgery and at several time-points during follow-up after surgery. The results showed that ≥1 CTC/30 ml of blood were detected in 44 (24%) patients before surgery. Patients with CTC before surgery had a significant decrease in recurrence-free survival (RFS, log-rank test p=0.014) and colon cancer related survival (CCRS, p=0.002). The 5-year RFS dropped from 75 to 61% and the 5-year CCRS from 83 to 69% for patients with CTC before surgery. The presence of CTC and positive lymph nodes remained significant factors in multivariate analysis for recurrence-free survival (RFS). Surprisingly, the presence of CTC weeks after surgery was not significantly associated with RFS and CCRD whereas CTC 2-3 years after surgery was again significantly associated with RFS and CCRD. The presence of CTC in patients with stage I-III CRC before surgery is associated with a significant reduction in RFS and CCRS. These findings suggest a role of CTC detection to assess which patients need adjuvant treatment.

Circulating tumor cells before and during follow-up after breast cancer surgery.

The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free and overall survival for patients with metastatic and newly diagnosed breast cancer. The present study was undertaken to explore whether the presence of CTC before and during follow-up after surgery is associated with recurrence free survival (RFS) and overall survival (OS). In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 403 stage I-III patients before undergoing surgery for breast cancer (A) and if available 1 week after surgery (B), after adjuvant chemo- and/or radiotherapy or before start of long-term hormonal therapy (C), one (D), two (E) and three (F) years after surgery. Patients were stratified into unfavorable (CTC≥1) and favorable (CTC=0) prognostic groups. >1 CTC in 30 ml blood was detected in 75/403 (19%) at A, 66/367 (18%) at B, 40/263 (15%) at C, 30/235 (12%) at D, 18/144 (11%) at E and 11/83 (13%) at F. RFS and OS was significantly lower for unfavorable CTC as compared to favorable CTC before surgery (p=0.022 and p=0.006), after adjuvant therapy (p<0.001 and p=0.018) and one (p=0.006 and p=0.013) and two (p<0.001 and p=0.045) years after surgery, but not 1 week post-surgery. The presence of CTC in blood drawn pre and one and two years after surgery, but not post-surgery is associated with shorter RFS and OS for stage I-III breast cancer.

Circulating tumor cells, disease recurrence and survival in newly diagnosed breast cancer.

INTRODUCTION: The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free survival and breast cancer-related death (BRD) for patients with metastatic breast cancer beginning a new line of systemic therapy. The current study was undertaken to explore whether the presence of CTC at the time of diagnosis was associated with recurrence-free survival (RFS) and BRD. METHODS: In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 602 patients before undergoing surgery for breast cancer. There were 97 patients with a benign tumor, 101 did not meet the inclusion criteria of which there were 48 patients with DCIS, leaving 404 stage I to III patients. Patients were stratified into unfavorable (CTC ≥1) and favorable (CTC = 0) prognostic groups. RESULTS: ≥1 CTC in 30 ml blood was detected in 15 (15%) benign tumors, in 9 DCIS (19%), in 28 (16%) stage I, 32 (18%) stage II and in 16 (31%) patients with stage III. In stage I to III patients 76 (19%) had ≥1 CTC of whom 16 (21.1%) developed a recurrence. In 328 patients with 0 CTC 38 (11.6%) developed a recurrence. Four-year RFS was 88.4% for favorable CTC and 78.9% for unfavorable CTC (P = 0.038). A total of 25 patients died of breast cancer-related causes and 11 (44%) had ≥1 CTC. BRD was 4.3% for favorable and 14.5% for unfavorable CTC (P = 0.001). In multivariate analysis ≥1 CTC was associated with distant disease-free survival, but not for overall recurrence-free survival. CTC, progesterone receptor and N-stage were independent predictors of BRD in multivariate analysis. CONCLUSIONS: Presence of CTC in breast cancer patients before undergoing surgery with curative intent is associated with an increased risk for breast cancer-related death.

External quality assurance of circulating tumor cell enumeration using the CellSearch(®) system: a feasibility study.

BACKGROUND: Circulating tumor cells (CTCs) are cells that have detached from solid tumors and entered the blood. CTCs can be detected, among others, by semi-automated immunomagnetic enrichment and image cytometry using CellSearch® (Veridex, Raritan, NJ). We studied the feasibility of external quality assurance (EQA) of the entire CellSearch procedure from blood draw to interpretation of results in multiple laboratories. METHODS: Blood samples from six cancer patients and controls were distributed to 14 independent laboratories to test between-laboratory, between-assay, and between-instrument variation. Additionally, between-operator variability was assessed through the interpretation of blinded images of all blood samples on a website. RESULTS: Shipment and storage of samples had no influence on CTC values. Between-instrument (coefficient of variation (CV) < 12%) and between-assay variation was low (CV ≤ 20%), indicating high reproducibility. However, between-laboratory CV ranged from 45 to 64%. Although inter-operator agreement on image interpretation (Fleiss' κ statistics) ranged from "substantial" to "almost perfect," image interpretation, particularly of samples containing high numbers of apoptotic cells, was the main contributor to between-laboratory variation. CONCLUSIONS: This multicenter study shows the feasibility of an EQA program for CTC detection in patient samples, and the importance of continuation of such a program for the harmonization of CTC enumeration.

CellTracks TDI: an image cytometer for cell characterization.

Characterization of rare cells usually requires high sensitivity quantification of multiple parameters. Detection of morphological features of these cells is highly desired when routinely identifying circulating tumor cells (CTC) in blood of patients. We have designed an image cytometer intended for fast and sensitive routine analysis of CTC. After an initial scan, prospective events can be revisited for more detailed analysis. The image cytometer features: 375, 491, and 639 nm laser lines, a 40×/0.6NA objective, a CCD camera operating in TDI mode, servo stages to move the sample in two dimensions and a piëzo microscope objective positioner to move the objective in the third dimension. ImageJ is used for dedicated image analysis. A homogeneous illumination area, measuring 180 × 180 µm(2) , was created by the use of a rotating diffuser in combination with two micro-lens arrays. For feed-forward automatic focusing of the sample during a scan, a 3D spline was fitted through 30 predetermined focus positions before scanning the sample. Continuous signal acquisition is made possible by using a CCD operating in TDI mode synchronized to the movement of two servo scan stages. The limit of fluorescence sensitivity is 120 PE molecules on a bead with a diameter of 6.8 µm, at a scanning speed of 1.0 mm s(-1) . The resolution of the imaging system is 0.76 µm in the TDI scan direction at a wavelength of 580 nm. Identification of cells is facilitated by scatter plots of the fluorescent parameters in which each individual event can be viewed for its morphological features by fluorescence as well as bright field. The image cytometer measures quantitative fluorescence and morphological features at a high sensitivity, high resolution, and with minimal overhead time. It has the ability torelocate events of interest for further detailed analysis. The system can be used for routine identification and characterization of rare cells.

Characterization of circulating tumor cells by fluorescence in situ hybridization.

Tumor cells in blood of patients with metastatic carcinomas have been associated with poor survival prospects. Further characterization of these cells may provide further insights into the metastatic process. Circulating Tumor Cells (CTC) were enumerated in 7.5 mL of blood with the CellSearch system. After enumeration of Cytokeratin+, CD45-, nucleated cells, the cells are fixed in the cartridge while maintaining their original position. Cartridges were hybridized with FISH probes against the centromeric regions of chromosome 1, 7, 8, and 17. Next fluorescence images of the FISH probes of the previous identified CTC were acquired. Leukocytes surrounding the CTC were used as internal controls. The number of copies of chromosome 1, 7, 8, and 17 could be determined in 118 CTC containing blood samples from 59 metastatic prostate cancer patients. The samples contained a total of 21,751 CTC (mean 184, median 16, SD 650). Chromosome counts were obtained in 61% of the relocated CTC. On an average, these CTC contained 2.8 copies of chromosome 1, 2.7 copies of chromosome 7, 3.1 copies of chromosome 8, and 2.3 copies of chromosome 17. CTC in which no chromosome count was obtained most likely underwent apoptosis indicated by the expression of M30. In 6/59 patients only diploid CTC were detected these samples, however, only contained 1-5 CTC. Heterogeneity in the chromosomal abnormalities was observed between CTC of different patients as well as among CTC of the same patient. Cytogenetic composition of CTC can be reliably assessed after they have been identified by the CellSearch system. The majority of CTC in hormone refractory prostate cancer are aneuploid confirming that they indeed are cancer cells. An extensive heterogeneity in the copy number of each of the chromosomes was observed.

CD4+ T lymphocytes enumeration by an easy-to-use single platform image cytometer for HIV monitoring in resource-constrained settings.

BACKGROUND: HIV monitoring in resource-constrained settings demands affordable and reliable CD4(+) T lymphocytes enumeration methods. We developed a simple single platform image cytometer (SP ICM), which is a dedicated volumetric CD4(+) T lymphocytes enumeration system that uses immunomagnetic and immunofluorescent technologies. The instrument was designed to be a low-cost, yet reliable and robust one. In this article we test the instrument and the immunochemical procedures used on blood from HIV negative and HIV positive patients. METHODS: After CD4 immunomagnetic labeling in whole blood, CD4(+) T lymphocytes, CD4(+dim) monocytes and some nonspecifically labeled cells are magnetically attracted to an analysis surface. Combining with CD3-Phycoerythrin (PE) labeling, only CD3(+)CD4(+) T lymphocytes are fluorescently labeled and visible in a fluorescent image of the analysis surface. The number of CD4(+) T lymphocytes is obtained by image analysis. Alternatively, CD3 immunomagnetic selection in combination with CD4 immunofluorescent labeling can also be applied for CD4(+) T lymphocytes enumeration. RESULTS: The SP ICM system was compared with two single platform flow cytometer (SP FCM) methods: tetraCXP and TruCount methods. The SP ICM system has excellent precision, accuracy and linearity for CD4(+) T lymphocytes enumeration. Good correlations were obtained between the SP ICM and the SP FCM methods for blood specimens of 44 HIV(-) patients, and of 63 HIV(+) patients. Bland-Altman plots showed interchangeability between the SP ICM and the SP FCM methods. CONCLUSIONS: The immunolabeling methods and the instrumentation are simple and easy-to-handle for less-trained operators. The SP ICM system is a good candidate for CD4(+) T lymphocytes enumeration in point-of-care settings of resource-constrained countries.

An immunomagnetic single-platform image cytometer for cell enumeration based on antibody specificity.

Simplification of cell enumeration technologies is necessary, especially for resource-poor countries, where reliable and affordable enumeration systems are greatly needed. In this paper, an immunomagnetic single-platform image cytometer (SP ICM) for cell enumeration based on antibody specificity is reported. A chamber/magnet assembly was designed such that the immunomagnetically labeled, acridine orange-stained cells in a blood sample moved to the surface of the chamber, where a fluorescent image was captured and analyzed for cell enumeration. The system was evaluated by applying one kind of antibody to count leukocytes and one kind for each leukocyte subpopulation: CD45 for leukocytes, CD3 for T lymphocytes, and CD19 for B lymphocytes. Excellent precision and linearity were achieved. Moreover, these cell counts, each from blood specimens of 42 to 52 randomly selected patients, were compared with those obtained by SP (TruCount) and dual-platform (DP) flow cytometry (FCM) technologies. The cell counts obtained by our system were in between those obtained from the TruCount and DP FCM methods; and good correlations were achieved (R > or = 0.95). For CD4(+) counts, as we expected, the cell count by our system was significantly higher than the CD4(+) T-lymphocyte counts obtained by SP and DP FCM methods. Immunophenotyping of the immunomagnetically selected CD4(+) cells showed that, besides CD4(+) T lymphocytes, a proportion of the CD4(+) dim monocytes was also selected. Our system is a simple immunomagnetic SP ICM, which can potentially be used for enumeration of CD3(+) CD4(+) T lymphocytes in resource-poor countries if an additional CD3 immunofluorescent label is applied.

A single platform image cytometer for resource-poor settings to monitor disease progression in HIV infection.

BACKGROUND: For resource-poor countries, affordable methods are required for enumeration of CD4(+) T lymphocytes of HIV-positive patients. For infants, additional determination of CD4/CD8 ratio is needed. METHODS: We determine the CD4(+) and CD8(+) T lymphocytes as the CD3(+)CD4(+) and CD3(+)CD8(+) population of blood cells. Target cells are CD3-immunomagnetically separated from the whole blood, and CD4-Phycoerythrin and CD8-PerCP immunofluorescently labeled. A point-of-care single platform image cytometer was developed to enumerate the target CD3(+)CD4(+) and CD3(+)CD8(+) populations. It has light-emitting diodes illumination, is fully computer-controlled, operates from a 12 V battery, and was designed to be cheap and easy-to-handle. Target cells are imaged on a CCD camera and enumerated by an image analysis algorithm. The cytometer outputs the absolute number of CD4(+) and CD8(+) T lymphocytes/microl and CD4/CD8 ratio. RESULTS: The quality of the cell images obtained with the cytometer is sufficient for a reliable enumeration of target cells. The image cytometer achieves an accuracy of better than 10% in the range of 50-1700 cells/microl. Analysis of blood samples from HIV patients yields a good agreement with the TruCount method for CD4 and CD8 count and CD4/CD8 ratio. CONCLUSIONS: The image cytometer is affordable (component costs $3,000), compact (25 x 25 x 20 cm(3)), and uses disposable test materials, making it a good candidate to monitor progression of immunodeficiency disease in resource-poor settings.

Statistical considerations for enumeration of circulating tumor cells.

BACKGROUND: Circulating tumor cells (CTCs) in patients with carcinomas are extremely rare. In metastatic breast cancer, the presence of >or=5 CTCs in 7.5 ml of blood has been associated with short survival. As this threshold has clinical implications, it is important to recognize the limitations associated with the detection and enumeration of CTCs. METHODS: Statistical analyses were performed on data generated from a multi-center clinical trial that utilized the CellSearchtrade mark System to isolate and enumerate CTCs in 7.5 ml blood samples. The statistical issues associated with each step of the process, from blood collection to final image analysis and CTC enumeration, were determined and implemented into a model. RESULTS: A model describing the statistics of the different process steps that are needed for the isolation and detection of CTCs was developed. The model uses the Poisson distribution for blood collection and empirically determined distributions for the isolation and identification of CTCs. The variability between readers was identified as one of the main sources of errors responsible for the current threshold level of five CTCs. CONCLUSIONS: Elimination of the errors made in the identification of tumor cells isolated from 7.5 ml of blood could potentially reduce the CTC threshold for the identification of patients with a poor prognosis from the current value of five CTCs to one CTC per 7.5 ml of blood.

Comparison of two methods for enumerating circulating tumor cells in carcinoma patients.

BACKGROUND: Monitoring of circulating tumor cells (CTCs) in blood of carcinoma patients treated with novel compounds may be a measurement of treatment effectiveness. Before it can be used clinically, a reliably method is needed to enumerate CTCs. We compared two methods for CTC enumeration, OnkoQuick and the CellSearch system. METHODS: We drew 22.5 ml of blood into three CellSave tubes from 15 healthy donors and 61 patients with metastatic carcinoma. After pooling, 15 ml was processed with OncoQuick and 7.5 ml with CellSearch. RESULTS: With both methods no CTCs were found in healthy donors. At least one CTC was detected in 14 of 61 patients (23%) with OncoQuick and 33 of 61 patients (54%) with CellSearch (P < 0.0001). The number of CTCs detected was larger for CellSearch (mean 20 CTCs/7.5 ml of blood) than for OncoQuick (3 CTCs/7.5 ml; P < 0.0001). CONCLUSION: The CellSearch system is a more accurate and sensitive method to enumerate CTCs. Further studies are warranted to evaluate CTC enumeration by the CellSearch system as a monitoring tool for the evaluation of the efficacy of novel anticancer agents.

Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.

PURPOSE: The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas. EXPERIMENTAL DESIGN: The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors. RESULTS: Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of >/=85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had >/=2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 +/- 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had >/=2 CTCs. Detection of >/=2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers. CONCLUSIONS: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.

Imaging technique implemented in CellTracks system.

BACKGROUND: We developed the CellTracks cell analysis system that, similar to flow cytometry, yields multiparameter information by which the cells can be differentiated. We describe the implementation of a laser scanning imaging method in the system. Image analysis of the cells improves the specificity of cell classification, especially in cases where the particular cells are found relatively infrequently and one has to discriminate between artifacts and real events. METHODS: Fluorescent images of immunomagnetically labeled and aligned cells are obtained by passing the cells through a laser focus. The laser focus is smaller than the objects and subsequent frames captured by a regular surveillance CCD camera with a frame grabber board represent different parts of the cells. Complete images of the cells are constructed by shifting each image with respect to each other and adding individual pixel values. RESULTS: The power of combining a fluorescent image with multiparametric data is demonstrated by imaging fluorescent and magnetically labeled beads and cells. The image gives additional information about the dye distribution across the objects. Changes in dye distribution as a function of time were observed in leukocytes labeled with the red fluorescent label, Oxazine750, which are imaged at different time intervals. CONCLUSIONS: An imaging technique implemented in the CellTracks system provides high-resolution fluorescent images of events previously identified by the system. The images of the fluorescent cells enhance the ability to classify rare events.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

BACKGROUND: Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. RESULTS: A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. CONCLUSIONS: The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension.