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.

"Platelet-associated regulatory system (PARS)" with particular reference to female reproduction.

BACKGROUND: Blood platelets play an essential role in hemostasis, thrombosis and coagulation of blood. Beyond these classic functions their involvement in inflammatory, neoplastic and immune processes was also investigated. It is well known, that platelets have an armament of soluble molecules, factors, mediators, chemokines, cytokines and neurotransmitters in their granules, and have multiple adhesion molecules and receptors on their surface. METHODS: Selected relevant literature and own views and experiences as clinical observations have been used. RESULTS: Considering that platelets are indispensable in numerous homeostatic endocrine functions, it is reasonable to suppose that a platelet-associated regulatory system (PARS) may exist; internal or external triggers and/or stimuli may complement and connect regulatory pathways aimed towards target tissues and/or cells. The signal (PAF, or other tissue/cell specific factors) comes from the stimulated (by the e.g., hypophyseal hormones, bacteria, external factors, etc.) organs or cells, and activates platelets. Platelet activation means their aggregation, sludge formation, furthermore the release of the for-mentioned biologically very powerful factors, which can locally amplify and deepen the tissue specific cell reactions. If this process is impaired or inhibited for any reason, the specifically stimulated organ shows hypofunction. When PARS is upregulated, organ hyperfunction may occur that culminate in severe diseases. CONCLUSION: Based on clinical and experimental evidences we propose that platelets modulate the function of hypothalamo-hypophyseal-ovarian system. Specifically, hypothalamic GnRH releases FSH from the anterior pituitary, which induces and stimulates follicular and oocyte maturation and steroid hormone secretion in the ovary. At the same time follicular cells enhance PAF production. Through these pathways activated platelets are accumulated in the follicular vessels surrounding the follicle and due to its released soluble molecules (factors, mediators, chemokines, cytokines, neurotransmitters) locally increase oocyte maturation and hormone secretion. Therefore we suggest that platelets are not only a small participant but may be the conductor or active mediator of this complex regulatory system which has several unrevealed mechanisms. In other words platelets are corpuscular messengers, or are more than a member of the family providing hemostasis.

Label-free, high-throughput, electrical detection of cells in droplets.

Today, droplet based microfluidics has become a standard platform for high-throughput single cell experimentation and analysis. However, until now no label-free, integrated single cell detection and discrimination method in droplets is available. We present here a microfluidic chip for fast (>100 Hz) and label-free electrical impedance based detection of cells in droplets. The microfluidic glass-PDMS device consists of two main components, the droplet generator and the impedance sensor. The planar electrode pair in the main channel allows the detection of only cells and cell containing droplets passing the electrodes using electrical impedance measurements. At a measurement frequency of 100 kHz non-viable cells, in low-conducting (LC) buffer, show an increase in impedance, due to the resistive effect of the membrane. The opposite effect, an impedance decrease, was observed when a viable cell passed the electrode pair, caused by the presence of the conducting cytoplasm. Moreover, we found that the presence of a viable cell in a droplet also decreased the measured electrical impedance. This impedance change was not visible when a droplet containing a non-viable cell or an empty droplet passed the electrode pair. A non-viable cell in a droplet and an empty droplet were equally classified. Hence, droplets containing (viable) cells can be discriminated from empty droplets. In conclusion, these results provide us with a valuable method to label-free detect and select viable cells in droplets. Furthermore, the proposed method provides the first step towards additional information regarding the encapsulated cells (e.g., size, number, morphology). Moreover, this all-electric approach allows for all-integrated Lab on a Chip (LOC) devices for cell applications using droplet-based platforms.

Microparticles and acute renal dysfunction in septic patients.

PURPOSE: The role of microparticles (MPs) in the pathogenesis of sepsis is not completely elucidated. We aimed to assess changes in the number of MPs during severe sepsis to follow the effect of sepsis-related organ failures, particularly renal impairment, an independent mortality factor of sepsis. MATERIALS AND METHODS: Thirty-seven severe septic patients and 20 controls were enrolled. Patient status as well as organ failure-related laboratory markers was followed up to 5 consecutive days. Microparticles (annexin V+ events in MP size gate) of platelet (CD41, CD42a, and PAC1), monocyte (CD14), and myeloid cell line (CD13) origin were measured using flow cytometry. RESULTS: Significantly increased total MP and CD41-, CD42a-, and PAC1-positive particle numbers were found in septic patients compared with controls. Actual number of organ dysfunctions on sample collection showed no correlation with MP numbers. Septic patients with renal dysfunction showed an increase in total MP, CD41(+), and CD13(+) particle numbers on admission. Amounts of platelet-derived CD42a(+) particles from patients with sepsis-related renal injury correlated negatively with actual blood urea nitrogen and creatinine concentrations. CONCLUSION: The increased numbers of platelet-derived MPs in severe septic patients emphasize the possible contribution of the hemostasis system in the development of sepsis-related renal impairments.

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.

Activated platelet-derived microparticle numbers are elevated in patients with severe fungal (Candida albicans) sepsis.

BACKGROUND: The treatment of severe sepsis highly depends on the identification of bacteria or fungi from blood and/or other body materials. Although widely available blood culturing and risk assessment scores are not completely reliable, current guidelines do not recommend the wide empirical use of antifungal medications based on questionable benefit or possible side-effects. We aimed to test whether platelet-derived microparticle (MP) measurements can improve the early detection of the infective agent behind sepsis. METHODS: Thirty-three consecutive severe septic patients from our university intensive care unit were included in our prospective study. MP number and surface antigen characteristics were followed by flow cytometry on days 1 (admission), 3 and 5. For microbiological identification, various specimens were collected on admission and in case of overall status deterioration. RESULTS: On admission, septic patients showed elevated annexin V and constitutive platelet marker (CD41)-positive MP numbers compared with volunteers. Mixed fungal septic patients showed significantly elevated annexin V and CD41-positive particle numbers on day 1 (P < 0.05) compared with the non-fungal septic group. Adhesive platelet marker (CD42a) harbouring vesicles were negligible in the non-fungal group, while fungal septic patients showed significantly elevated numbers in all measurements (P < 0.01). Particles from activated platelets (PAC1) had elevated numbers in the first and fifth study days compared with non-fungal septic patients (P < 0.05). CONCLUSIONS: The measurement of CD42a- and PAC1-positive microparticles may provide important additional information which can help to improve the early instalment of antifungal therapy of severe septic patients.

Steroidogenesis in the adrenal dysfunction of critical illness: impact of etomidate.

INTRODUCTION: This study was aimed at characterizing basal and adrenocorticotropic hormone (ACTH)-induced steroidogenesis in sepsis and nonsepsis patients with a suspicion of critical illness-related corticosteroid insufficiency (CIRCI), taking the use of etomidate-inhibiting 11ß-hydroxylase into account. METHOD: This was a prospective study in a mixed surgical/medical intensive care unit (ICU) of a university hospital. The patients were 62 critically ill patients with a clinical suspicion of CIRCI. The patients underwent a 250-µg ACTH test (n = 67). ACTH, adrenal steroids, substrates, and precursors (modified tandem mass spectrometry) also were measured. Clinical characteristics including use of etomidate to facilitate intubation (n = 14 within 72 hours of ACTH testing) were recorded. RESULTS: At the time of ACTH testing, patients had septic (n = 43) or nonseptic critical illness (n = 24). Baseline cortisol directly related to sepsis and endogenous ACTH, independent of etomidate use. Etomidate was associated with a lower baseline cortisol and cortisol/11ß-deoxycortisol ratio as well as higher 11ß-deoxycortisol, reflecting greater 11ß-hydroxylase inhibition in nonsepsis than in sepsis. Cortisol increases < 250 mM in exogenous ACTH were associated with relatively low baseline (HDL-) cholesterol, and high endogenous ACTH with low cortisol/ACTH ratio, independent of etomidate. Although cortisol increases with exogenous ACTH, levels were lower in sepsis than in nonsepsis patients, and etomidate was associated with diminished increases in cortisol with exogenous ACTH, so that its use increased, albeit nonsignificantly, low cortisol increases to exogenous ACTH from 38% to 57%, in both conditions. CONCLUSIONS: A single dose of etomidate may attenuate stimulated more than basal cortisol synthesis. However, it may only partly contribute, particularly in the stressed sepsis patient, to the adrenal dysfunction of CIRCI, in addition to substrate deficiency.

High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel.

In this article high-yield (77%) and high-speed (2700 cells s(-1)) single cell droplet encapsulation is described using a Dean-coupled inertial ordering of cells in a simple curved continuous microchannel. By introducing the Dean force, the particles will order to one equilibrium position after travelling less than 1 cm. We use a planar curved microchannel structure in PDMS to spatially order two types of myeloid leukemic cells (HL60 and K562 cells), enabling deterministic single cell encapsulation in picolitre drops. An efficiency of up to 77% was reached, overcoming the limitations imposed by Poisson statistics for random cell loading, which yields only 37% of drops containing a single cell. Furthermore, we confirm that > 90% of the cells remain viable. The simple planar structure and high throughput provided by this passive microfluidic approach makes it attractive for implementation in lab on a chip (LOC) devices for single cell applications using droplet-based platforms.

Future Prospects in Breast Cancer Research - Cancer Stem Cells.

Breast cancer is one of the leading causes of cancer deaths among women. Although significant advances in the prevention, diagnosis and management are made, still every year half a million women die of breast cancer. Personalised treatment has the potential to increase treatment efficacy, and hence decrease mortality rates. Moreover, understanding cancer biology and translating this knowledge to the clinic, will improve the breast cancer therapy regime tremendously. Recently, it has been proposed that cancer stem cells (CSC) play an important role in tumour biology. CSC have the ability for self-renewal and are pivotal in setting the heterogeneous character of a tumour. Additionally, CSC possess several characteristics that make them resistant and more aggressive to the conventional chemo- and radiotherapy. Nowadays, breast cancer therapy is focused on killing the differentiated tumour cells, leaving the CSC unharmed, potentially causing recurrence of the disease and metastasis. Specific targeting of the CSC will improve the disease-free survival of breast cancer patients. In this article, two methods are described, aiming at specifically attacking the differentiated tumour cells ('Apoptosis chip') and the cancer stem cell. For this, microfluidics is used.

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation.

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 µs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.

Evaluation of tubular poly(trimethylene carbonate) tissue engineering scaffolds in a circulating pulsatile flow system.

Tubular scaffolds (internal diameter approximately 3 mm and wall thickness approximately 0.8 mm) with a porosity of approximately 83% and an average pore size of 116 µm were prepared from flexible poly(trimethylene carbonate) (PTMC) polymer by dip-coating and particulate leaching methods. PTMC is a flexible and biocompatible polymer that crosslinks upon irradiation; porous network structures were obtained by irradiating the specimens in vacuum at 25 kGy before leaching soluble salt particles. To assess the suitability of these scaffolds in dynamic cell culturing for cardiovascular tissue engineering, the scaffolds were coated with a thin (0.1 to 0.2 mm) non-porous PTMC layer and its performance was evaluated in a closed pulsatile flow system (PFS). For this, the PFS was operated at physiological conditions at liquid flows of 1.56 ml/s with pressures varying from 80-120 mmHg at a frequency of 70 pulsations per minute. The mechanical properties of these coated porous PTMC scaffolds were not significantly different than non-coated scaffolds. Typical tensile strengths in the radial direction were 0.15 MPa, initial stiffness values were close to 1.4 MPa. Their creep resistance in cyclic deformation experiments was excellent. In the pulsatile flow setup, the distention rates of these flexible and elastic scaffolds were approximately 0.10% per mmHg, which is comparable to that of a porcine carotid artery (0.11% per mmHg). The compliance and stiffness index values were close to those of natural arteries.?In long-term deformation studies, where the scaffolds were subjected to physiological pulsatile pressures for one week, the morphology and mechanical properties of the PTMC scaffolds did not change. This suggests their suitability for application in a dynamic cell culture bioreactor.

A new floating electrode structure for generating homogeneous electrical fields in microfluidic channels.

In this article a new parallel electrode structure in a microfluidic channel is described that makes use of a floating electrode to get a homogeneous electrical field. Compared to existing parallel electrode structures, the new structure has an easier production process and there is no need for an electrical connection to both sides of the microfluidic chip. With the new chip design, polystyrene beads suspended in background electrolyte have been detected using electrical impedance measurements. The results of electrical impedance changes caused by beads passing the electrodes are compared with results in a similar planar electrode configuration. It is shown that in the new configuration the coefficient of variation of the impedance changes is lower compared to the planar configuration (0.39 versus 0.56) and less dependent on the position of the beads passage in the channel as a result of the homogeneous electrical field. To our knowledge this is the first time that a floating electrode is used for the realization of a parallel electrode structure. The proposed production method for parallel electrodes in microfluidic channels can easily be applied to other applications.

Dynamic culturing of smooth muscle cells in tubular poly(trimethylene carbonate) scaffolds for vascular tissue engineering.

Porous, tubular, flexible, and elastic poly(trimethylene carbonate) (PTMC) scaffolds (length 8 cm and inner diameter 3 mm) for vascular tissue engineering were prepared by means of a dip-coating and particulate leaching procedure. Using NaCl as porogen, scaffolds with an average pore size of 110 µm and a porosity of 85% were obtained. Before leaching the salt, the structures were made creep-resistant by means of crosslinking at 25 kGy gamma irradiation. To increase the efficiency of cell seeding, the scaffolds were provided with a microporous outer layer of 0.2 mm with an average pore size of 28 µm and a porosity of 65% (total wall thickness 1 mm). Human smooth muscle cells (SMCs) were seeded in these scaffolds with an efficiency of 43%, as determined after 24 h cell adhesion. SMCs were cultured in the scaffolds up to 14 days under stationary conditions or under pulsatile flow conditions in a bioreactor (pressure 70-130 mmHg, 69 pulsations/min, and average wall shear rate 320 s(-1)). Although SMCs proliferated under both conditions, cell numbers were three to five times higher in case of dynamic culturing. This was qualitatively confirmed by means of histology. Also, in terms of mechanical properties, the dynamically cultured constructs performed better than the statically cultured constructs. After culturing for 14 days, the maximum tensile strengths of the constructs, determined in the radial direction, had increased from 0.16 MPa (unseeded scaffold) to 0.48 MPa (dynamic culturing) and 0.38 MPa (static culturing). The results of this study indicate that a potentially useful medial layer for tissue-engineered vascular grafts can be prepared by dynamic culturing of human SMCs seeded in porous tubular poly(trimethylene carbonate) scaffolds.

Cancer prevalence in osteoporotic women with low serum vitamin D levels.

OBJECTIVE: The aim of this study was to assess the role of vitamin D in cancer development in postmenopausal osteoporotic women. METHODS: A cross-sectional and in vitro study was carried out, with statistical analysis with odds ratios and 95% CIs presented. Human estrogen receptor-positive breast cancer cells (MCF-7) were studied in vitro. The apoptosis-to-proliferation (A/P) ratio was also determined. RESULTS: A total of 885 women were included in this study. Any kind of cancer was found in 112 (12.7%) of all women. Breast cancer was the most prevalent malignancy, representing half of the cases (n = 56, 50%). The prevalence of any kind of cancer and breast cancer in women with low 25-hydroxyvitamin D3 levels (25OHD; <50 nmol/L) was higher than in women with high 25OHD levels (≥ 50 nmol/L). The in vitro study demonstrated a statistically significant increased A/P ratio of 5.27 (95% CI, 4.054-6.493) with a high concentration of 1α,25-dihydroxyvitamin D (10 µM) after 96 hours. CONCLUSIONS: Osteoporotic women with low serum levels of 25OHD (<50 nmol/L) have an increased prevalence of any kind of cancer and breast cancer; however, these differences are not statistically significant. 1α,25-dihydroxyvitamin D induced an increased A/P ratio in MCF-7 breast cancer cells in vitro.

On-chip determination of spermatozoa concentration using electrical impedance measurements.

In this article we describe the development of a microfluidic chip to determine the concentration of spermatozoa in semen, which is a main quality parameter for the fertility of a man. A microfluidic glass-glass chip is used, consisting of a microchannel with a planar electrode pair that allows the detection of spermatozoa passing the electrodes using electrical impedance measurements. Cells other than spermatozoa in semen also cause a change in impedance when passing the electrodes, interfering with the spermatozoa count. We demonstrate that the change in electrical impedance is related to the size of cells passing the electrodes, allowing to distinguish between spermatozoa and HL-60 cells suspended in washing medium. In the same way we are able to distinguish between polystyrene beads and spermatozoa. Thus, by adding a known concentration of polystyrene beads to a boar semen sample, the spermatozoa concentrations of seven mixtures are measured and show a good correlation with the actual concentration (R(2)-value = 0.97). To our knowledge this is the first time that the concentration of spermatozoa has been determined on chip using electrical impedance measurements without a need to know the actual flow speed. The proposed method to determine the concentration can be easily applied to other cells. The described on-chip determination of the spermatozoa concentration is a first step towards a microfluidic system for a complete quality analysis of semen.

A microfluidic wound-healing assay for quantifying endothelial cell migration.

Endothelial migration is an important process in the formation of blood vessels and the repair of damaged tissue. To study this process in the laboratory, versatile and reliable migration assays are essential. The purpose of this study was to investigate whether the microfluidic version of the conventional wound-healing assay is a useful research tool for vascular science. Endothelial cells were seeded in a 500-mum-wide microfluidic channel. After overnight incubation, cells had formed a viable and confluent monolayer. Then, a wound was generated in this monolayer by flushing the channel with three parallel fluid streams, of which the middle one contained the protease trypsin. By analyzing the closing of the wound over time, endothelial cell migration could be measured. Although the migration rate was two times lower in the microfluidic assay than in the conventional assay, an identical 1.5-times increase in migration rate was found in both assays when vascular endothelial growth factor (VEGF(165)) was added. In the microfluidic wound-healing assay, a stable gradient of VEGF(165) could be generated at the wound edge. This led to a two-times increase in migration rate compared with the untreated control. Finally, when a shear stress of 1.3 Pa was applied to the wound, the migration rate increased 1.8 times. In conclusion, the microfluidic assay is a solid alternative for the conventional wound-healing assay when endothelial cell migration is measured. Moreover, it offers unique advantages, such as gradient generation and application of shear stress.

Apoptosis- and necrosis-induced changes in light attenuation measured by optical coherence tomography.

Optical coherence tomography (OCT) was used to determine optical properties of pelleted human fibroblasts in which necrosis or apoptosis had been induced. We analysed the OCT data, including both the scattering properties of the medium and the axial point spread function of the OCT system. The optical attenuation coefficient in necrotic cells decreased from 2.2 +/- 0.3 mm(1) to 1.3 +/- 0.6 mm(-1), whereas, in the apoptotic cells, an increase to 6.4 +/- 1.7 mm(-1) was observed. The results from cultured cells, as presented in this study, indicate the ability of OCT to detect and differentiate between viable, apoptotic, and necrotic cells, based on their attenuation coefficient. This functional supplement to high-resolution OCT imaging can be of great clinical benefit, enabling on-line monitoring of tissues, e.g. for feedback in cancer treatment.

Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs.

Breast cancer is the leading cause of cancer deaths among non-smoking women worldwide. At the moment the treatment regime is such that patients receive different chemotherapeutic and/or hormonal treatments dependent on the hormone receptor status, the menopausal status and age. However, in vitro sensitivity testing of tumor biopsies could rationalize and improve the choice of chemo- and hormone therapy. Lab-on-a-Chip devices, using microfluidic techniques, make detailed cellular analysis possible using fewer cells, enabling working with a patients' own cells and performing chemo- and hormone sensitivity testing in an ex vivo setting. This article describes the development of two microfluidic devices made in poly(dimethylsiloxane) (PDMS) to validate the cell culture properties and analyze the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine (SSP). In both cases, cell viability was assessed using the life-stain Calcein-AM (CAAM) and the death dye propidium iodide (PI). MCF-7 cells could be statically cultured for up to 7 days in the microfluidic chip. A 30 min flow with SSP and a subsequent 24 h static incubation in the incubator induced apoptosis in MCF-7 cells, as shown by a disappearance of the aggregate-like morphology, a decrease in CAAM staining and an increase in PI staining. This work provides valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells in response to therapeutics and in this way improve cancer treatment towards personalized medicine.

Controlled cavitation-cell interaction: trans-membrane transport and viability studies.

Cavitation bubble dynamics close to a rigid surface gives rise to a rapid and transient fluid flow. A single bubble is created with a laser pulse at different stand-off distances from the rigid surface, where the stand-off distance gamma is defined by gamma = h/R(max), with h being the initial distance and R(max) being the maximum bubble radius. When the surface is covered with adherent cells, molecular delivery and cell detachment after single cavitation activity are observed at different locations. We find a maximum of cell detachment at a normalized stand-off distance of gamma approximately 0.65. In contrast, the maximum of the molecular uptake is found when gamma approaches 0. The single cavitation event has only little effect on the viability of cells in the non-detached area. We find apoptosis of cells only very close to the area of detachment and, additionally, the metabolism of the non-detached cells shows no pronounced difference compared to control cells according to an MTS assay. Thus, although the cavitation event is responsible for the detachment of cells, only few of the remaining cells undergo a permanent change.

Microchip capillary electrophoresis for point-of-care analysis of lithium.

BACKGROUND: Microchip capillary electrophoresis (CE) is a promising method for chemical analysis of complex samples such as whole blood. We evaluated the method for point-of-care testing of lithium. METHODS: Chemical separation was performed on standard glass microchip CE devices with a conductivity detector as described in previous work. Here we demonstrate a new sample-to-chip interface. Initially, we took a glass capillary as a sample collector for whole blood from a finger stick. In addition, we designed a novel disposable sample collector and tested it against the clinical standard at the hospital (Medisch Spectrum Twente). Both types of collectors require < 10 microL of test fluid. The collectors contain an integrated filter membrane, which prevents the transfer of blood cells into the microchip. The combination of such a sample collector with microchip CE allows point-of-care measurements without the need for off-chip sample treatment. This new on-chip protocol was verified against routine lithium testing of 5 patients in the hospital. RESULTS: Sodium, lithium, magnesium, and calcium were separated in < 20 s. The detection limit for lithium was 0.15 mmol/L. CONCLUSIONS: The new microchip CE system provides a convenient and rapid method for point-of-care testing of electrolytes in serum and whole blood.