Replacement of specific markers for apoptosis and necrosis by nuclear morphology for affordable cytometry.

OBJECTIVE: The objective of the present study was to employ high throughput image analysis to detect necrosis and apoptosis. Specific markers were replaced by morphological parameters of cells and nuclei. METHOD: Fresh blood was taken from a healthy female and given a treatment to induce cell necrosis and apoptosis. Afterward, the samples were stained with AnnexinV-FITC, DRAQ5 and DAPI. Slides were made and analyzed using the cytometer iCys. Pictures were scanned. The analyzed sample consisted of 73 sets of images of DAPI, DRAQ5 and AnnexinV-FITC, respectively. For image analysis and subsequent statistical processing, the CellProfiler and CellProfilerAnalyst were used. Each sample was analyzed twice. The first analysis was conducted using the markers (DAPI, DRAQ5 and Annexin) for an unequivocal identification and subsequent count of necrotic, apoptotic and live cells (gold standard). Thereafter, a second analysis was performed for the nuclear morphology and texture (morphometric analysis). After the machine learning process was completed, the software calculated the quantity of cells in each of the three groups. A comparison between the result of the gold standard and the morphometric analysis was performed using linear regression and a Bland-Altman test. RESULTS: The linear regression between the two compared analyses was r(2)=0.57 for apoptosis, r(2)=0.84 for necrosis and r(2)=0.79 for living cells. CONCLUSION: It may be concluded that it is possible to replace specific markers against morphology without losing the reproducible high-throughput character of a cytometric analysis.

Agonist-induced ß2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells.

It is not clear whether increased asthma severity associated with long-term use of ß2-adrenoceptor (ß2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between ß-AR agonist-mediated effects on ß2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o(-) cells grown in the presence and absence of ß-AR agonists and/or antagonists, the ß2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in ß2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the ß2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered ß2-AR expression and function in airway epithelial cells. ß2-AR desensitization and downregulation induced by long-term treatment with ß2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Morphometry to identify subtypes of leukocytes.

INTRODUCTION: Recent studies in image cytometry evaluated the replacement of specific markers by morphological parameters. The aim of this study was to develop and evaluate a method to identify subtypes of leukocytes using morphometric data of the nuclei. METHOD: The analyzed images were generated with a laser scanning cytometer. Two free programs were used for image analysis and statistical evaluation: Cellprofiler and Tanagra respectively. A sample of leukocytes with 200 sets of images (DAPI, CD45 and CD14) was analyzed. Using feature selection, the 20 best parameters were chosen to conduct cross-validation. RESULTS: The morphometric data identified the subpopulations of the analyzed leukocytes with a sensitivity and specificity of 0.95 per sample. CONCLUSION: The present study is the first that identifies subpopulations of leukocytes by nuclear morphology.

An innovative cascade system for simultaneous separation of multiple cell types.

Isolation of different cell types from one sample by fluorescence activated cell sorting is standard but expensive and time consuming. Magnetic separation is more cost effective and faster by but requires substantial effort. An innovative pluriBead-cascade cell isolation system (pluriSelect GmbH, Leipzig, Germany) simultaneously separates two or more different cell types. It is based on antibody-mediated binding of cells to beads of different size and their isolation with sieves of different mesh-size. For the first time, we validated the pluriSelect system for simultaneous separation of CD4+- and CD8+-cells from human EDTA-blood samples. Results were compared with those obtained by magnetic activated cell sorting (MACS; two steps -first isolation of CD4+, then restaining of the residual cell suspension with anti-human CD8+ MACS antibody followed by the second isolation). pluriSelect separation was done in whole blood, MACS separation on density gradient isolated mononuclear cells. Isolated and residual cells were immunophenotyped by 7-color 9-marker panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLA-DR) using flow cytometry. Cell count, purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (MACS (median[range]: 92.4% [91.5-94.9] vs. pluriSelect 95% [94.9-96.8])) of CD4+ cells, however CD8+ isolation showed lower purity by MACS (74.8% [67.6-77.9], pluriSelect 89.9% [89.0-95.7]). Yield was not significantly different for CD4 (MACS 58.5% [54.1-67.5], pluriSelect 67.9% [56.8-69.8]) and for CD8 (MACS 57.2% [41.3-72.0], pluriSelect 67.2% [60.0-78.5]). Viability was slightly higher with MACS for CD4+ (98.4% [97.8-99.0], pluriSelect 94.1% [92.1-95.2]) and for CD8+-cells (98.8% [98.3-99.1], pluriSelect 86.7% [84.2-89.9]). pluriSelect separation was substantially faster than MACS (1h vs. 2.5h) and no pre-enrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two and more cell subpopulation directly from whole blood and provides a simple alternative to magnetic separation.

Label-free hybridoma cell culture quality control by a chip-based impedance flow cytometer.

Impedance flow cytometry (IFC) was evaluated as a possible alternative to fluorescence-based methods for on-line quality monitoring of hybridoma cells. Hybridoma cells were cultured at different cell densities and viability was estimated by means of IFC and fluorescence-based flow cytometry (FCM). Cell death was determined by measuring the impedance phase value at high frequency in low conductivity buffer. IFC data correlate well with reference FCM measurements using AnnexinV and 7-AAD staining. Hybridoma cells growing at different densities in cell culture revealed a density-dependent subpopulation pattern. Living cells of high density cultures show reduced impedance amplitudes, indicating particular cellular changes. Dead cell subpopulations become evident in cultures with increasing cell densities. In addition, a novel intermediate subpopulation, which most probably represents apoptotic cells, was identified. These results emphasize the extraordinary sensitivity of high frequency impedance measurements and their suitability for hybridoma cell culture quality control.

Changes in neuronal DNA content variation in the human brain during aging.

The human brain has been proposed to represent a genetic mosaic, containing a small but constant number of neurons with an amount of DNA exceeding the diploid level that appear to be generated through various chromosome segregation defects initially. While a portion of these cells apparently die during development, neurons with abnormal chromosomal copy number have been identified in the mature brain. This genomic alteration might to lead to chromosomal instability affecting neuronal viability and could thus contribute to age-related mental disorders. Changes in the frequency of neurons with such structural genomic variation in the adult and aging brain, however, are unknown. Here, we quantified the frequency of neurons with a more than diploid DNA content in the cerebral cortex of normal human brain and analyzed its changes between the fourth and ninth decades of life. We applied a protocol of slide-based cytometry optimized for DNA quantification of single identified neurons, which allowed to analyze the DNA content of about 500 000 neurons for each brain. On average, 11.5% of cortical neurons showed DNA content above the diploid level. The frequency of neurons with this genomic alteration was highest at younger age and declined with age. Our results indicate that the genomic variation associated with DNA content exceeding the diploid level might compromise viability of these neurons in the aging brain and might thus contribute to susceptibilities for age-related CNS disorders. Alternatively, a potential selection bias of "healthy aging brains" needs to be considered, assuming that DNA content variation above a certain threshold associates with Alzheimer's disease.

Identification and evaluation of novel synovial tissue biomarkers in rheumatoid arthritis by laser scanning cytometry.

INTRODUCTION: Suitable biomarkers are essential for therapeutic strategies in personalized medicine in terms of diagnosis as well as of prognosis. With highly specific biomarkers, it is possible, for example, to identify patients with poor prognosis, which enables early intervention and intensive treatment. The aim of this study was to identify and validate biomarkers and possible combinations for a prospective use in immunoscintigraphy, which may improve diagnosis of rheumatoid arthritis (RA) patients with consideration of inflammatory activity in the affected joints. Therefore, we tested several monoclonal antibodies (mAbs) directed against cellular-surface molecules on cells likely to be involved in the pathogenesis of RA. METHODS: Synovial tissue from patients with long-standing RA (accompanied by synovitis with varying states of current activity) and patients with acute non-RA arthritis were stained for surface molecules on different cell types by using fluorochrome-labeled antibodies. Tissue analysis was done by laser scanning cytometry (LSC), and statistical evaluation, by discriminant analysis and ROC analysis. RESULTS: CD11b, HLA-DR, CD90, and CD64 revealed significant differences between tissues from patients with RA and acute non-RA arthritis. Especially with the expression of CD64, both patient cohorts could be discriminated with high sensitivity and specificity. RA classification was improved by simultaneously investigating the expression of two or three different surface proteins, such as HLA-DR, CD90, and CD29 in the tissue. The simultaneous analysis of CD64 together with CD304 or the combination of CD11b and CD38 was suitable for the identification of RA patients with high current activity in synovitis. CONCLUSIONS: In this study, we showed that LSC is a novel reliable method in biomarker prevalidation in RA. Hence, identified mAbs in situ may allow their potential use in in vivo approaches. Moreover, we proved that biomarker-combination analysis resulted in better discrimination than did single-marker analysis. Combinations of these markers make a novel and reliable panel for the discrimination between RA and acute non-RA arthritis. In addition, further expedient combinations may be novel promising biomarker panels to identify current activity in synovitis in RA.

Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR-inhibitors in peripheral human blood.

BACKGROUND: Therapeutic drug monitoring (TDM) of immunosuppressive drugs after organ transplantation is based on measuring blood levels alone, which often results in under- or over-immunosuppression. Previous studies have shown the potential of measuring pharmacodynamic drug effects for TDM, but assessment of biomarkers for individual drugs is still not clinical routine. Therefore, we validated a specific assay to measure the pharmacodynamic effects of mammalian target of rapamycin (mTOR)-inhibitors on phosphorylated S6 ribosomal protein (p-S6RP), a downstream target of mTOR. METHODS: Clinical relevant concentrations of sirolimus (SRL, 0.9-91.4 µg/L), cyclosporine A (CsA, 75.1-1202 µg/L), mycophenolate acid (MPA, 0.08-3.2 mg/L), or dexamethasone (DEX, 0.5-200 ng/mL) were added to whole-blood from healthy volunteers. Activated whole-blood was analyzed by phospho-flow cytometry to measure p-S6RP in T cells. RESULTS: Phospho-flow analysis revealed that SRL suppressed p-S6RP in human T cells in a dose-dependent manner with a half-maximal inhibitory concentration (IC(50)) at 19.8 nM and a maximal inhibitory effect (I(max) %) at 91.9%. Neither CsA, MPA, nor DEX inhibited mTOR-related S6RP-phosphorylation. Coefficient of variations from 0.03 to 0.05, 0.12 to 0.25, and 0.14 to 0.38 for intra-, interassay, and interindividual variability respectively, showed robustness of our assay. Furthermore, samples can be stored at RT or 4°C up to 2 h after withdrawal. CONCLUSION: We validated a robust whole-blood assay that allows the specific measurement of SRL- and everolimus-induced inhibition of T cells' function through detection of p-S6RP. Future studies in organ transplanted recipients will show if this assay has the potential to enhance a TDM for mTOR-inhibitor drugs in combination therapies.

Recent advances in cytometry applications: preclinical, clinical, and cell biology.

The acceptance of flow cytometry (FCM) in clinical laboratory medicine is a major stepping stone towards development new cell analyses, improvement of accuracy, and finally a new range of diagnostic tests. Applications range from differential blood count determination to the identification of fluorescence-labeled subpopulations of disease-specific cell types in cell suspensions. Even new disease patterns can be identified by FCM. However, FCM is not only applicable for making a diagnosis but also for disease monitoring and routine check-ups. It is often used in oncology-related analyses, such as for leukemia and lymphoma patients. Here, not only cell numbers are relevant but also the degree of antigen expression which can be determined in a standardized way. Next to FCM also image cytometry has entered clinical applications although manual review by pathologists is still standard. In general, the multicolor approach and hence the ability for multiparametric analyses has led FCM to a central cornerstone in cell biology research. This review is intended to present an overview of cytometric applications which have entered clinical practice and led to deeper understanding in biological processes.

Introduction A: recent advances in cytometry instrumentation, probes, and methods--review.

Cytometric techniques are continually being improved, refined, and adapted to new applications. This chapter briefly outlines recent advances in the field of cytometry with the main focus on new instrumentations in flow and image cytometry as well as new probes suitable for multiparametric analyses. There is a remarkable trend for miniaturizing cytometers, developing label-free and fluorescence-free analytical approaches, and designing "intelligent" probes. Furthermore, new methods for analyzing complex data for extracting relevant information are reviewed.

On the quantification of intracellular proteins in multifluorescence-labeled rat brain slices using slide-based cytometry.

In several brain regions, a subpopulation of neurons exists being characterized by the expression of a peculiar form of extracellular matrix, a so-called perineuronal net (PNN). We have previously shown that the PNN can bind large amounts of iron due to its polyanionic charge. Because free iron can generate reactive oxygen species thus being potentially toxic, the PNN may have a protective function by "scavenging" this free iron. Because of this ability, we have hypothesized that PNN-related neurons have an altered iron-specific metabolism. Thus, to compare the intracellular concentrations of iron containing proteins, specifically, the iron storage protein ferritin H between neurons with and without a PNN, we have used slide-based cytometry with image-based threshold-boundary cell detection on brain sections. In tissue sections, the integrity of the extracellular matrix, especially the characteristic PNNs, is preserved, which is necessary for the identification of the two neuronal subpopulations. A multilabeling approach was chosen to select neurons (neuronal marker NeuN), to classify the neurons according to their subtype (matrix marker Wisteria floribunda agglutinin), and to quantify the protein concentration (protein marker). Using this novel method, we were able to detect a relative difference in protein concentration as low as 12% between the two subpopulations of neurons in the neuronal population of the rat parietal cortex.

Cellular analysis by open-source software for affordable cytometry.

Image cytometry is an important technique in affordable healthcare and cellular research. Some efforts toward establishing a personal, low-cost cytometer have been described in the literature. However, a self-assembled fluorescence microscope requires software for cytometric analysis. There are some open-source image-based software analysis applications available. However, for a quantitative analysis of images, software that can generate data comparable to those of previously evaluated cytometric analyses programs is required. Hence, the aim of this study is to compare results of a commercially available image cytometry program to data obtained using the open-source software CellProfiler (CP). Leukocytes and fluorescent bead images obtained using a Laser Scanning Cytometer were analyzed by CP and the results compared with those of conventional cytometric analyses' programs. Algorithms were developed enabling the analysis of leukocytes and beads by CP. CP provided similar results to those obtained by the cytometer software. Hallmark parameters, including cell count and fluorescence intensity, revealed a high correlation in the analysis of both programs. Therefore, CP is appropriate for cellular analysis on a self-assembled microscope, thereby enabling affordable cytometry.

PACAP up-regulates the expression of apolipoprotein D in 3T3-L1 adipocytes. DRG/3T3-L1 co-cultures study.

The existence of a cross-talk between nerves and fatty tissue is increasingly recognized. Using co-cultures of dorsal root ganglion (DRG)-derived cells and 3T3-L1 adipocytes, we have previously shown that the presence of fat cells enhances neurite outgrowth and number of synapses. Vice versa, neural cells induced expression of neurotrophic adipokines apolipoprotein D and E (ApoD, ApoE) and angiopoietin-1 (Ang-1) by adipocytes. Here, we tested whether pituitary adenylate cyclase-activating peptide (PACAP), which is released by sensory fibres and causes Ca(2+) influx into fat cells, is involved in ApoD induction. Using 3T3-L1 cell cultures, we found that PACAP at a dose of 1 nM up-regulated the expression of ApoD protein and mRNA approx. 2.5 fold. This effect was driven by ERK1/2 acting upon PAC1/VPAC2 receptors. In turn, PACAP-treated 3T3-L1 adipocytes in co-cultures with DRG cells enhanced neurite ramification of neurofilament 200 (NF200)-positive neurons (measured using fluorescence microscopy) and neurofilament 68 protein levels (measured using Western blot analysis). This effect could be blocked using the PAC1/VPAC2 antagonist PACAP(6-38). Scanning cytometry revealed PACAP/ApoD induced low density lipoprotein receptors (LDLR) and ApoE receptor 2 (apoER2) in NF200-positive cells. Thus, a bidirectional loop seems to exist regulating the innervation of fatty tissues: PACAP released from sensory fibres might stimulate fat cells to synthesize neurotrophic adipokines, which, in turn, support peripheral innervation.

Microfluidic impedance-based flow cytometry.

Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact.

Differences in the kinetics of gamma-H2AX fluorescence decay after exposure to low and high LET radiation.

PURPOSE: In order to obtain more insight into heavy ion tumour therapy, some features of the underlying molecular mechanisms controlling the cellular response to high linear energy transfer (LET) radiation are currently analysed. MATERIALS AND METHODS: We analysed the decay of the integrated fluorescence intensity of gamma-H2AX (phosphorylated histone H2AX) which is thought to reflect the repair kinetics of radiation-induced DNA double-strand breaks (DSB) using Laser-Scanning-Cytometry. Asynchronous human HeLa cells were irradiated with a single dose of either 1.89 Gy of 55 MeV carbon ions or 5 Gy of 70 kV X-rays. RESULTS: Measurements of the gamma-H2AX-intensities from 15-60 min resulted in a 16 % decrease for carbon ions and in a 43 % decrease for X-rays. After 21 h, the decrease was 77 % for carbon ions and 85 % for X-rays. The corresponding time-effect relationship was fitted by a bi-exponential function showing a fast and a slow component with identical half-life values for both radiation qualities being 24 +/- 4 min and 13.9 +/- 0.7 h, respectively. Apparent differences in the kinetics following high and low LET irradiation could completely be attributed to quantitative differences in their contributions, with the slow component being responsible for 47 % of the repair after exposure to X-rays as compared to 80 % after carbon ion irradiation. CONCLUSION: gamma-H2AX loss kinetics follows a bi-exponential decline with two definite decay times independent of LET. The higher contribution of the slow component determined for carbon ion exposure is thought to reflect the increased amount of complex DSB induced by high LET radiation.

Basics of standardization and calibration in cytometry--a review.

Standardization, calibration, and controls (negative and positive controls) are essential for quality assurance. Cytometers are capable of reliable and repeatable cellular analyses. However, a prerequisite is instrument calibration and standardized preanalytics. Calibration is often done by beads. Beads are available for different quality control applications, e.g. calibration of size and measuring scale, compensation, absolute cell counting, and laser alignment. Results can be standardized by converting MFI values into MESF or ABC values. Standardized data allow comparison of experiments over a long period of time and between different instruments and laboratories. Alterations in the sensitivity of the cytometer can be detected by routinely performing quality control. The process of quality assurance quantifies and helps manage the variance from the desired value. Results can thus be compared objectively with those of other laboratories. Standardization is the basis of cytometry and a prerequisite for obtaining reliable data.

Cytomics and nanobioengineering.

The finding that an individual's genome differs as much as by many million variants from that of the human reference assembly diminished the great enthusiasm that every disease could be predicted based on nucleotide polymorphisms. Even individual cells of an organ may be specifically equipped to perform specific tasks and that the information of individual cells in a cell system is key information to understand function or dysfunction. Therefore, cytomics received great attention during the last years as it allows to quantitatively and qualitatively analyzing great number of individual cells, cell constituents, and of their intracellular and functional interactions in a cellular system and also giving the concept of analysis of these data.Exhaustive data extraction from multiparametric assays and multiple tests are the prerequisite for prediction of drug toxicity. Cytomics, as novel approach for unsupervised data analysis give a chance to find the most predictive parameters, which describe best the toxicity of a chemical. Cytomics is intrinsically connected to drug development and drug discovery.Focused on small structures, nanobioengineering is the ideal partner of cytomics, the systems biological discipline for cell population analysis. Realizing the idea "from the molecule to the patient" develops and offers chemical compounds, proteins, and other biomolecules, cells as well as tissues as instruments and products for a wide variety of biotechnological and biomedical applications.The integrative nanobioengineering combining different disciplines of nanotechnology will promote the development of innovative therapies and diagnostic methods. It can improve the precision of the measurements with focus on single cell analysis. By nanobioengineering and whole body imaging techniques, cytomics covers the field from molecules through bacterial cells, eukaryotic tissues, and organs to small animal live analysis. Toxicological testing and medical drug development are currently strongly broadening. It harbors the promise to substantially impact on various fields of biomedicine, drug discovery, and predictive medicine.As the number of scientific data is rising exponentially, new data analysis tools and strategies like cytomics and nanobioengineering take a lead and get closer to application. Bionanoengineering may strongly support the quantitative data supply, thus strengthening the rational for cytomics approach.

Aneuploidy and DNA replication in the normal human brain and Alzheimer's disease.

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to approximately 10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of approximately 2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.