A sample-preparation-free, automated, sample-to-answer system for cell counting in human body fluids.

While many clinical laboratory tests are now highly automated, body fluid cell counting, particularly in low-cellularity samples such as cerebral spinal fluid (CSF), is often performed manually. Here, we report a simple, cost-effective method to obtain white and red blood cell counts from human body fluids such as CSF. The method consists of a compact, automated, and low-cost fluorescence microscope system, coupled to a sample chamber containing all of the necessary reagents in dry form to stain and prepare the sample. Sample focus and scanning are handled automatically, and the acquired multimodal images are automatically analyzed to extract cell counts. Comparison with manual counting on over 200 clinical samples shows excellent agreement. As the system counts a substantially larger image region than a standard manual cell count, we find our sensitivity to extremely low cellularity samples to potentially be higher than the manual gold standard, evidenced by our system recording images of cells in samples whose cell count was registered as "0" by a trained user. Thus, our system holds promise for routine, automated, and sensitive analysis of body fluids whose cellularity extends across a wide dynamic range.

Hematopoietic progenitor cell counting can optimize peripheral blood stem cell apheresis process.

INTRODUCTION: Monitoring of stem cell concentration in transplantation settings is crucial to determine the optimal time of apheresis and is currently based on enumeration of CD34+ cells by flow cytometry. No surrogate marker has replaced CD34+ cell enumeration to date. The aim of this study was the evaluation of the hematopoietic progenitor cell (HPC) parameter of the Sysmex XN-1000 analyzer in terms of optimizing the peripheral blood stem cell apheresis process. MATERIALS AND METHODS: Results of flow cytometric CD34+ cell and Sysmex HPC count were compared in 208 preharvest samples and 139 apheresis products. RESULTS: HPC and CD34+ cell counts showed significant differences in the multiple myeloma (MM) group. The correlation between preharvest HPC and CD34+ cell counts was good in the MM group (rho = .613) and strong in the lymphoma group (rho = .802), allogeneic donors (rho = .923), and other group of samples (rho = .816). The HPC positive cutoff demonstrating 100% specificity and positive predictive value for MM patients was high for ≥20/µL and ≥10/µL CD34+ cell counts, and therefore of limited value. The HPC negative cutoff demonstrating 100% sensitivity and negative predictive value was approximately <4/µL, irrespective of diagnosis. CONCLUSIONS: Based on proposed HPC positive cutoffs (≥31/µL in the lymphoma group and ≥11/µL in the other group of samples), routine HPC enumeration could improve the workflow by replacing CD34+ cell counting in allogeneic donors as well as non-MM patients. Furthermore, based on proposed HPC negative cutoff (<4/µL), CD34+ cell counting could be fully omitted in donors and patients that are not adequately mobilized.

The relationship of bovine milk somatic cell count to neutrophil level in samples of cow's milk assessed by an automatic cell counter.

This research communication describes the application of a fluorescent automatic cell counter Lactoscan SCC for simultaneous determination of somatic cell count and neutrophils in bovine milk. The obtained results were compared with results obtained by a flow cytometer and a light microscope. The Pearson correlations between the methods were calculated. A comparison between the main characteristics of the three kinds of analysis was made - the assay duration and the intra-assay precision. A relation between the SCC and neutrophil cells was observed in 55 milk samples. The obtained results confirm that the simultaneous determination of SCC and neutrophil analysis are necessary and support the early diagnosis of mastitis, the timely treatment of the animal and the avoidance of major economic losses.

Evaluation of urine dipsticks for quality control of residual erythrocytes and leukocytes in leukocyte-depleted donor plasma.

Currently used methodologies for quality control of residual leukocytes and erythrocytes in leukocyte-depleted plasma are either expensive or time-consuming. It has been proposed that urine dipsticks could be used as a screening method for residual erythrocytes. The aim was, therefore, to evaluate if urine dipsticks could be used to detect residual erythrocytes and also residual leukocytes in leukocyte-depleted plasma. Dilution series ranging over the decision limits for residual erythrocytes and leukocytes were prepared. Positive, negative and overall agreements, as well as the precision and joint frequency distributions, were calculated for five dipstick analyzers and their corresponding dipsticks. Twenty-four consecutive leukocyte-depleted donor plasma samples were also tested. None of the dipstick analyzers had both a high positive and a high negative agreement. Accordingly, none of the analyzers were able to discriminate between cell concentrations close to the decision limits. The inconsistency count revealed differences in precision between the dipstick analyzers. In the 24 consecutive donor samples, no significant correlation between the dipstick analyzers and the reference methods were found. In conclusion, urine dipsticks are not suitable for quality control of residual leukocytes and erythrocytes in leukocyte-depleted donor plasma.

Improvement of cell counting method for Neubauer counting chamber.

BACKGROUND: We compared the cell counting accuracy of the conventional method and the improved method by using Neubauer counting chamber. METHODS: In the improved method, all the border cells were counted and then divided by two; while, in the conventional method, only border cells on the two boundaries (top and left) were counted. RESULTS: About 55.814% of the samples showed more accurate results by improved counting method, about 38.372% had more accurate results by conventional counting method, and about 5.814% were counted with similar counting error by both methods. The improved method had significantly smaller counting error than conventional method (P < .05). The distribution ratio of the border cells was an independent factor for counting accuracy (P < .05). CONCLUSION: Together, the improved counting method can reduce the counting error of the Neubauer counting chamber to some extent, assess the distributing uniformity of border cells, and help to eliminate the samples with large differences in distribution.

A portable microfluidic platform for rapid determination of microbial load and somatic cell count in milk.

Microfluidics systems that have been emerged in the last 20 years and used for processing the fluid in a microchannel structure at microliter levels are alternative to the conventional methods. The objective of the study is to develop a microfluidic platform for determination of the microbial load and the number of somatic cells in milk. For this purpose, a polydimethylsiloxane (PDMS) chip with a channel size of 300 µm × 60 µm was produced. Cells/bacteria labeled with fluorescent stain in milk were counted with the proposed microfluidic platform and the results were compared with the reference cell concentration/the bacterial counts by conventional method. It was found that our platform could count somatic and bacterial cells with an accuracy above 80% in 20 min run for each analysis. The portable overall platform has an overall dimension of 25x25x25 cm and weighs approximately 9 kg.

Molecular Profiling of Pooled Circulating Tumor Cells from Prostate Cancer Patients Using a Dual-Antibody-Functionalized Microfluidic Device.

To capture both epithelial and mesenchymal subpopulations of CTCs at different metastatic stages of PCa patients, here we constructed a novel dual-antibody-functionalized microfluidic device by employing antibodies against PSMA and EpCAM. In vitro experiments with the dual capture system for capturing both LnCAP and LnCAP-EMT cells have shown significantly enhanced capture efficiency as compared to that of the EpCAM single capture system. Furthermore, the dual capture system could successfully identify CTCs in 20 out of 24 (83.3%) PCa patients, and the CTCs counts from the dual capture system were statistically correlated with the TNM stage of patients ( P < 0.05), while conventional diagnostic methods, such as serum PSA level and Gleason score, failed to correlate to patient TNM stages. To further explore potential clinical application of our dual capture system, captured CTCs were recovered and subjected to qRT-PCR to quantify known factors involved in PCa development and therapy. The results demonstrated that the combined detection of SChLAP1 and PSA in CTCs is a potential marker for identifying patients with metastatic PCa, while detection of AR and PD-L1 in CTCs may have the potential to determine the sensitivity of PCa patients to androgen deprivation therapy and immunotherapy, respectively. Taken together, the dual-antibody-functionalized microfluidic device established in our study overcomes the limitations of some CTC capture platforms that only detect epithelial or mesenchymal CTCs in PCa patients, and detection of the PCa-related RNA signatures from purified CTCs holds great promise to offer warnings for early metastasis of PCa and may provide guidance for therapy decisions.

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PURPOSE: To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. METHODS: B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. RESULTS: All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). CONCLUSION: FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Performance Evaluation of a New Generation of Automated Analyzer for Pleural and Peritoneal Fluids Cytology.

BACKGROUND: Hematologic counters with dedicated body fluids mode allows these samples to be analyzed in an automated manner. Our aim was to evaluate the performance of the Sysmex XN-3000 and compare it with the Sysmex XE-5000, in use in our laboratory for cytological analysis of body fluids. METHODS: We studied 108 pleural and peritoneal fluids. Laboratory routine included manual and automated cell counts. The Sysmex XN-3000 validation protocol included precision, carryover, linearity studies, and comparison with traditional microscopic differential counts and with the analyzer in use. RESULTS: Sysmex XN-3000 met all the criteria for analytical quality with strong correlation with microscopy (r = 0.95 for MN and PMN) and agreement of 93% (kappa = 0.813, p < 0.0001). Comparison between both analyzers revealed no significant differences and strong correlation regarding WBC and RBC (r > 0.98), mononuclear and polymorphonuclear cells (r = 0.99). CONCLUSIONS: Sysmex XN-3000 showed strong correlation and agreement with traditional microscopy with an equivalent performance compared to the XE-5000.

Suppression Technique of HeLa Cell Proliferation Using Ultrasonic Power Amplifiers Integrated with a Series-Diode Linearizer.

A series-diode linearizer scheme is developed, which can possibly generate higher voltage signals. To verify our proposed concept, ultrasonic power amplifiers with and without the linearizer were tested for HeLa cells proliferation in vitro. In general, ultrasonic stimulus initiates the process of cavitation which can cause cell lysis and disruption of cell attachment. The cavitation can also induce formation of free radicals so that a rigid membrane of malignant cancer cells have increased sensitivity to ultrasonic stimulus. The cell density of the control group increased up to almost 100% on Day 3. However, cell densities of the experimental group when using an isolated ultrasonic power amplifier, and ultrasonic power amplifiers integrated with the linearizer at 1 V and 5 V DC (direct current) bias could be suppressed more than that when using an ultrasonic power amplifier (90.7 ± 1.2%, 75.8 ± 3.5%, and 68.1 ± 1.1%, respectively). Additionally, the proliferation suppressing ratios of each experimental group confirmed that the cell density decrements of the experimental groups exhibited statistical significance compared to the control group (ultrasonic power amplifier = 8.87%, ultrasonic power amplifier with 1 V biased linearizer = 23.87%, and ultrasonic power amplifier with 5 V biased linearizer = 31.56%).

Performance of the XN-2000 WPC channel-flagging to differentiate reactive and neoplastic leukocytosis.

BACKGROUND: The distinction between reactive and neoplastic leukocytes, especially atypical lymphocytes suspected to be reactive or neoplastic, is a particular challenge in automated hematological cell differentiation. The aim of the study was to evaluate the performance of the XN analyzer supplemented with the WPC channel for differentiating between reactive and neoplastic leukocytosis. METHODS: Blood samples of 253 patients with viral infections, lymphoma or leukemia were analyzed by the Sysmex XN-2000 analyzer equipped with the WPC channel. The results were compared to routine leukocyte differentiation using the routine Sysmex XE-2100 analyzer and automated digital microscopy (DM96). The combined information from standard morphology, immune phenotyping and clinical diagnosis served as a reference. RESULTS: The XN WPC channel demonstrated an excellent performance for differentiating neoplastic (AUC=0.933) and reactive leukocytosis (AUC=0.900) as compared to morphological smear examination (AUC=0.949 and AUC=0.968, respectively) or to the differentiation results of our routine hematology analyzer (AUC=0.630 and AUC=0.635, respectively). CONCLUSIONS: Our data show that the combined WDF/WPC of the Sysmex XN-Series analyzer is advantageous in the automated differentiation of neoplastic and reactive leukocytosis, thus supporting the correct diagnostic decision in the daily laboratory routine.

Effects of rapid decompression on HUVECs in vitro.

OBJECTIVE: To explore the possible effects of rapid decompression on the activity and function of vascular endothelial cells in vitro. METHODS: Human umbilical vein endothelial cell (HUVEC) cultures were exposed at 7 atmospheres absolute (atm abs) air for two hours before decompression. Two decompression profiles were used at the rate of 30 atm abs min-1 (rapid decompression) or 1 atm abs min-1 (normal decompression). Three hours after decompression, cell activity was detected by cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) activity assay; cell permeability was measured by electrical resistance determinations. Twelve hours after decompression, cell apoptosis was detected by flow cytometry with Annexin V FITC/PI double staining. RESULTS: There was no significant statistical difference between rapid and normal decompression groups in all the determined parameters (P=0.59, 0.87, 0.86 and 0.81, respectively). CONCLUSIONS: HUVECs endure rapid decompression well from 7 atm abs at the rate of 30 atm abs min-1, or the current determinations are not sensitive enough to reveal the possible injuries. Further research with more sensitive indexes is warranted to reveal the possible effects and mechanisms.

How to count cells: the advantages and disadvantages of the isotropic fractionator compared with stereology.

The number of cells comprising biological structures represents fundamental information in basic anatomy, development, aging, drug tests, pathology and genetic manipulations. Obtaining unbiased estimates of cell numbers, however, was until recently possible only through stereological techniques, which require specific training, equipment, histological processing and appropriate sampling strategies applied to structures with a homogeneous distribution of cell bodies. An alternative, the isotropic fractionator (IF), became available in 2005 as a fast and inexpensive method that requires little training, no specific software and only a few materials before it can be used to quantify total numbers of neuronal and non-neuronal cells in a whole organ such as the brain or any dissectible regions thereof. This method entails transforming a highly anisotropic tissue into a homogeneous suspension of free-floating nuclei that can then be counted under the microscope or by flow cytometry and identified morphologically and immunocytochemically as neuronal or non-neuronal. We compare the advantages and disadvantages of each method and provide researchers with guidelines for choosing the best method for their particular needs. IF is as accurate as unbiased stereology and faster than stereological techniques, as it requires no elaborate histological processing or sampling paradigms, providing reliable estimates in a few days rather than many weeks. Tissue shrinkage is also not an issue, since the estimates provided are independent of tissue volume. The main disadvantage of IF, however, is that it necessarily destroys the tissue analyzed and thus provides no spatial information on the cellular composition of biological regions of interest.

Self-calibrating highly sensitive dynamic capacitance sensor: towards rapid sensing and counting of particles in laminar flow systems.

In this report we propose a sensor architecture and a corresponding read-out technique on silicon for the detection of dynamic capacitance change. This approach can be applied to rapid particle counting and single particle sensing in a fluidic system. The sensing principle is based on capacitance variation of an interdigitated electrode (IDE) structure embedded in an oscillator circuit. The capacitance scaling of the IDE results in frequency modulation of the oscillator. A demodulator architecture is employed to provide a read-out of the frequency modulation caused by the capacitance change. A self-calibrating technique is employed at the read-out amplifier stage. The capacitance variation of the IDE due to particle flow causing frequency modulation and the corresponding demodulator read-out has been analytically modelled. Experimental verification of the established model and the functionality of the sensor chip were shown using a modulating capacitor independent of fluidic integration. The initial results show that the sensor is capable of detecting frequency changes of the order of 100 parts per million (PPM), which translates to a shift of 1.43 MHz at 14.3 GHz operating frequency. It is also shown that a capacitance change every 3 µs can be accurately detected.

International study on inter-reader variability for circulating tumor cells in breast cancer.

INTRODUCTION: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. METHODS: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. RESULTS: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). CONCLUSIONS: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

Measuring cone density in a Japanese macaque (Macaca fuscata) model of age-related macular degeneration with commercially available adaptive optics.

The aim of this study was to assess the feasibility of using a commercially available high-resolution adaptive optics (AO) camera to image the cone mosaic in Japanese macaques (Macaca fuscata) with dominantly inherited drusen. The macaques examined develop drusen closely resembling those seen in humans with age-related macular degeneration (AMD). For each animal, we acquired and processed images from the AO camera, montaged the results into a composite image, applied custom cone-counting software to detect individual cone photoreceptors, and created a cone density map of the macular region. We conclude that flood-illuminated AO provides a promising method of visualizing the cone mosaic in nonhuman primates. Future studies will quantify the longitudinal change in the cone mosaic and its relationship to the severity of drusen in these animals.

Measuring single-cell density.

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL(-1). We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes.

Application of an online-biomass sensor in an optical multisensory platform prototype for growth monitoring of biotechnical relevant microorganism and cell lines in single-use shake flasks.

In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae [1]. It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This sensor based measurement method has a high potential to initiate a new generation of online bioprocess monitoring. Metabolic studies will particularly benefit from the multisensory data acquisition. The sensor is already used in labscale experiments for shake flask cultivations.

An automated high-throughput counting method for screening circulating tumor cells in peripheral blood.

Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel, and interrogated by a line-confocal microscope. On the basis of the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 min. We applied this method in analyzing CTCs from 90 stage IV breast cancer patient samples and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by the U.S. Food and Drug Administration at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n = 9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast cancer patients ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast cancer patients that were positive for Her2 or CD44(+)/CD24(-), which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.

Cell diameter measurements obtained with a handheld cell counter could be used as a surrogate marker of G2/M arrest and apoptosis in colon cancer cell lines exposed to SN-38.

In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancer cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the (3)H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.