Assessment of goblet cell size and density in relation to epithelial cell (multi)layering on conjunctival impression cytology samples.

PURPOSE: To assess goblet cell size and numbers in relation to the extent of multilayering of conjunctival impression cytology (CIC) samples as a basis for reducing variability in image selection for goblet cell density (GCD) estimates. METHODS: CIC was undertaken immediately postmortem off the superior bulbar conjunctiva of healthy young adult rabbits onto Millicell-CM Biopore filter units. After fixation with buffered glutaraldehyde and Giemsa staining, two × 200 images were selected from each sample representative of either slight multilayering or substantial multilayering, projected at × 1000, an overlay of the outlines of the goblet cells was made, and their dimensions and areas were measured. RESULTS: From measures of 4918 goblet cells, the average value (+/- SD) for the longest dimension was 17.7 ± 6.4 µm and 14.6 ± 5.3 µm for the shortest dimension. The GCD values ranged from 210 to 2069/mm2, with a mean of 1074 ± 601/mm2, but was lower for slightly multilayered images (at 537 ± 239 cells/mm ) compared with multilayered regions (at 1612 ± 601 cells/mm2; p < 0.001). The measured areas ranged from 72 to 491 µm2, with average values from any particular image ranging from 110 to 370 µm2, which were inversely correlated with the estimated GCD (Spearman's rho = - 0.722, p < 0.05). CONCLUSIONS: Larger goblet cells but in fewer numbers were predictably found across the filter surface where there were fewer layers of cells and vice versa. This difference could be considered in selection of images for counts of goblet cells from CIC specimens.

A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential.

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.

A rapid cell-based assay for determining poloxamer quality in CHO suspension cell culture.

Poloxamers are water-soluble polymers that are widely used in cell culture bioprocessing to protect cells against shearing forces. Use of poor-quality poloxamers may lead to a drastic reduction in cell growth, viabilities and productivities in cell culture-based manufacturing. In order to evaluate poloxamer quality and promote more consistent performance, a rapid cell membrane adhesion to hydrocarbon assay was developed based on the adhesive properties of cell membranes to selective hydrocarbons. The assay can identify a poor-performing poloxamer characterized by significant drop in viable cell density and percent viability. The assay was verified across multiple good and bad poloxamer lots, and the results were in agreement with established cell growth and high-performance liquid chromatography assays.

QuickCount®: a novel automated software for rapid cell detection and quantification.

We describe a novel automated cell detection and counting software, QuickCount® (QC), designed for rapid quantification of cells. The Bland-Altman plot and intraclass correlation coefficient (ICC) analyses demonstrated strong agreement between cell counts from QC to manual counts (mean and SD: -3.3 ± 4.5; ICC = 0.95). QC has higher recall in comparison to ImageJauto, CellProfiler and CellC and the precision of QC, ImageJauto, CellProfiler and CellC are high and comparable. QC can precisely delineate and count single cells from images of different cell densities with precision and recall above 0.9. QC is unique as it is equipped with real-time preview while optimizing the parameters for accurate cell count and needs minimum hands-on time where hundreds of images can be analyzed automatically in a matter of milliseconds. In conclusion, QC offers a rapid, accurate and versatile solution for large-scale cell quantification and addresses the challenges often faced in cell biology research.

Summary of the National Institute of Standards and Technology and US Food And Drug Administration cell counting workshop: Sharing practices in cell counting measurements.

The emergence of cell-based therapeutics has increased the need for high-quality, robust and validated measurements for cell characterization. Cell count, being one of the most fundamental measures for cell-based therapeutics, now requires increased levels of measurement confidence. The National Institute of Standards and Technology (NIST) and the US Food and Drug Administration (FDA) jointly hosted a workshop focused on cell counting in April 2017 entitled "NIST-FDA Cell Counting Workshop: Sharing Practices in Cell Counting Measurements." The focus of the workshop was on approaches for selecting, designing and validating cell counting methods and overcoming gaps in obtaining sufficient measurement assurance for cell counting. Key workshop discussion points, representing approximately 50 subject matter experts from industry, academia and government agencies, are summarized here. A key conclusion is the need to design the most appropriate cell counting method, including control/measurement assurance strategies, for a specific counting purposes. There remains a need for documentary standards for streamlining the process to develop, qualify and validate cell counting measurements as well as community-driven efforts to develop new or improved biological and non-biological reference materials.

Relationship between monocytes to lymphocytes ratio and axial spondyloarthritis.

BACKGROUND: Axial spondyloarthritis (axSpA) is a progressive, chronic, inflammatory skeletal disorder affecting the spine and sacroiliac joints. Many studies have shown that neutrophils, lymphocytes, monocytes, platelets, and red blood cells (RBCs) play important roles in the inflammatory process of axSpA. Neutrophils to lymphocytes ratio (NLR) and red blood cell distribution width (RDW) have been reported to be simple and inexpensive markers to indicate the disease activity of axSpA. However, the role of monocytes to lymphocytes ratio (MLR) and platelets to lymphocytes ratio (PLR) in axSpA was rarely mentioned. OBJECTIVE: The study's aim was to determine the role of MLR and PLR in axSpA patients and to investigate their relationships with disease severity. METHODS: AxSpA patients who fulfilled the Assessment in Ankylosing Spondylitis International Society classification criteria published in 2009 were enrolled in this study and divided into nonradiographic axial spondyloarthritis (nr-axSpA) group and ankylosing spondylitis (AS) group. Healthy age and gender-matched subjects were also enrolled as control group. MLR, PLR, NLR, RDW, C-reactive protein (CRP) level, and erythrocyte sedimentation rate (ESR) level were assessed. The correlation between the variables with finger-to-floor distance, Modified Schober test, and occiput-to-wall distance were tested with Pearson correlation. Furthermore, area under curve (AUC) value, sensitivity, specificity, and the optimal cutoff values were determined using receiver operating characteristic (ROC) curves. RESULTS: A total of 148 axSpA patients (67 nr-axSpA patients and 81 AS patients) and 58 healthy subjects were included in the study. The MLR, NLR, PLR, and RDW in axSpA group were higher than those in the control group (P < 0.05). Among them, MLR and RDW were highly increased in AS group compared with the nr-axSpA group (P < 0.05). MLR, NLR, PLR, and RDW were all positively correlated with ESR level and CRP level (P < 0.05). MLR and RDW were positively correlated with finger-to-floor distance and negatively correlated with Modified Schober test (P < 0.05). RDW was positively correlated with occiput-to-wall distance (P < 0.05). ROC curve results showed MLR yielded a higher AUC than NLR, PLR, and RDW (P < 0.05). In addition, the optimal cutoff value of MLR for axSpA was 0.22, with a specificity of 70.9% and sensitivity of 68.4%. CONCLUSIONS: MLR was elevated in AS patients compared to nr-axSpA patients and had a close relationship with CRP level, ESR level, and spine movements. MLR may be a reliable, cost-effective, and novel potential parameter to evaluate disease severity in axSpA.

A segmentation method based on HMRF for the aided diagnosis of acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: The diagnosis of acute myeloid leukemia (AML) is purely dependent on counting the percentages of blasts (>20%) in the peripheral blood or bone marrow. Manual microscopic examination of peripheral blood or bone marrow aspirate smears is time consuming and less accurate. The first and very important step in blast recognition is the segmentation of the cells from the background for further cell feature extraction and cell classification. In this paper, we aimed to utilize computer technologies in image analysis and artificial intelligence to develop an automatic program for blast recognition and counting in the aspirate smears. METHODS: We proposed a method to analyze the aspirate smear images, which first performs segmentation of the cells by k-means cluster, then builds cell image representing model by HMRF (Hidden-Markov Random Field), estimates model parameters through probability of EM (expectation maximization), carries out convergence iteration until optimal value, and finally achieves second stage refined segmentation. Furthermore, the segmentation results are compared with several other methods using six classes of cells respectively. RESULTS: The proposed method was applied to six groups of cells from 61 bone marrow aspirate images, and compared with other algorithms for its performance on the analysis of the whole images, the segmentation of nucleus, and the efficiency of calculation. It showed improved segmentation results in both the cropped images and the whole images, which provide the base for down-stream cell feature extraction and identification. CONCLUSIONS: Segmentation of the aspirate smear images using the proposed method helps the analyst in differentiating six groups of cells and in the determination of blasts counting, which will be of great significance for the diagnosis of acute myeloid leukemia.

A Prospective Observational Study for Assessment and Outcome Association of Circulating Endothelial Cells in Clear Cell Renal Cell Carcinoma Patients Who Show Initial Benefit from First-line Treatment. The CIRCLES (CIRCuLating Endothelial cellS) Study (SOGUG-CEC-2011-01).

BACKGROUND: Markers able to predict the response to antiangiogenics in metastatic clear cell renal cell carcinoma (ccRCC) are not available. The development of new treatment options like immunotherapy are reaching the clinic; therefore, predictors of benefit from these different available treatments are increasingly needed. OBJECTIVE: In this study, we prospectively assessed the association of circulating endothelial cells (CECs) in peripheral blood with long-term benefit from first-line treatment in ccRCC. DESIGN, SETTING, AND PARTICIPANTS: A prospective observational study was designed involving 13 institutions of the Spanish Oncology Genitourinary Group. Adult patients diagnosed with advanced ccRCC who had achieved response or disease stabilization after 3 mo on first-line therapy were eligible. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: CECs were isolated from peripheral blood, captured with ferrofluids coated with monoclonal antibodies directed against the CD146 antigen, and assessed centrally with an automated standardized system. CECs were defined as 4',6-diamidino-2-phenylindole+, CD105+, and CD45-. Blood samples were systematically taken every 6 wk for 15 mo or until tumor progression, whichever occurred first. Clinical data were externally monitored at all centers. RESULTS AND LIMITATIONS: From August 9, 2011, to January 17, 2013, 75 patients were enrolled in the study. Patients with baseline CECs above the median showed a significantly longer progression-free survival than those with low CECs (22.2 mo vs 12.2 mo) with a hazard ratio of 2.5 (95% confidence interval: 1.2-5.3, p=0.016). There was no difference between CEC levels at baseline and at tumor progression (medians of 50 CECs/4ml and 52 CECs/4ml, respectively). CONCLUSIONS: Under antiangiogenic treatment, the detection of higher CEC levels is associated with clinical benefit in terms of progression-free survival in ccRCC. PATIENT SUMMARY: Antiangiogenics are the cornerstone of treatment in kidney cancer. Since they target endothelial rather than tumor cells, we studied the correlation between levels of circulating endothelial cells in peripheral blood and long-term benefit in patients on antiangiogenic therapy. Higher levels were associated with long-term benefit, suggesting that this determination could help to separate best responders from those who could require a more intensive approach.

Label-free high temporal resolution assessment of cell proliferation using digital holographic microscopy.

Cell proliferation assays are widely applied in biological sciences to understand the effect of drugs over time. However, current methods often assess cell population growth indirectly, that is, the cells are not actually counted. Instead other parameters, for example, the amount of protein, are determined. These methods often also demand phototoxic labels, have low temporal resolution, or employ end-point assays, and frequently are labor intensive. We have developed a robust and label-free kinetic cell proliferation assay with high temporal resolution for adherent cells using digital holographic microscopy (DHM), one of many quantitative phase microscopy techniques. As no labels or stains are required, and only very low intensity illumination is necessary, the technique allows for noninvasive continuous cell counting. Only two image processing settings were adjusted between cell lines, making the assay practical, user friendly, and free of user bias. The developed direct assay was validated by analyzing cell cultures treated with various concentrations of the anti-cancer drug etoposide, a well-established topoisomerase inhibitor that causes DNA damage and leads to programmed cell death. After treatment, the unstained adherent cells were nondestructively imaged every 30 min for 36 h inside a cell incubator. In the recorded time-lapse image sequences, individual cells were automatically identified to provide detailed growth curves and growth rate data of cell number, confluence, and average cell volume. Our results demonstrate how these parameters facilitate a deeper understanding of cell processes than what is achievable with current single-parameter and end-point methods. © 2017 International Society for Advancement of Cytometry.

Adequately defining tumor cell proportion in tissue samples for molecular testing improves interobserver reproducibility of its assessment.

Gene mutation status assessment of tumors has become standard practice in diagnostic pathology. This is done using samples comprising tumor cells but also non-tumor cells, which may dramatically dilute the proportion of tumor DNA and induce false negative results. Increasing sensitivity of molecular tests presently allows detection of a targeted mutation in a sample with a small percentage of tumor cells, but assessment of tumor cellularity remains essential to adequately interpret the results of molecular tests. Comprehensive tumor cell counting would provide the most reliable approach but is time consuming, and therefore rough global estimations are used, the reliability of which has been questioned in view of their potential clinical impact. The French association for quality assurance in pathology (AFAQAP) conducted two external quality assurance schemes, partly in partnership with the French group of oncology cytogenomics (GFCO). The purpose of the schemes was to (1) evaluate how tumor cellularity is assessed on tissue samples, (2) identify reasons for discrepancies, and (3) provide recommendations for standardization and improvement. Tumor cell percentages in tissue samples of lung and colon cancer were estimated by 40-50 participants, on 10 H&E virtual slides and 20 H&E conventional slides. The average difference between lowest and highest estimated percentage was 66. This was largely due to inadequate definition of cellularity, reflecting confusion between the percentage of tumor cells and the percentage of the area occupied by tumor in the assessed region. The widest range of interobserver variation was observed for samples with dense or scattered lymphocytic infiltrates or with mucinous stroma. Estimations were more accurate in cases with a low percentage of tumor cells. Macrodissection of the most homogeneous area in the tissue reduced inter-laboratory variation. We developed a rating system indicating potential clinical impact of a discrepancy. Fewer discrepancies were clinically relevant since the study was conducted. Although semi-quantitative estimations remain somewhat subjective, their reliability improves when tumor cellularity is adequately defined and heterogeneous tissue samples are macrodissected for molecular analysis.

Assessment of a rapid liquid-based cytology method for measuring sputum cell counts.

Differential sputum cell counting is not widely available despite proven clinical utility in the management of asthma. We compared eosinophil counts obtained using liquid-based cytology (LBC), a routine histopathological processing method, and the current standard method. Eosinophil counts obtained using LBC were a strong predictor of sputum eosinophilia (≥3%) determined by the standard method suggesting LBC could be used in the management of asthma.

Fully Automated Islet Cell Counter (ICC) for the Assessment of Islet Mass, Purity, and Size Distribution by Digital Image Analysis.

For isolated pancreatic islet cell preparations, it is important to be able to reliably assess their mass and quality, and for clinical applications, it is part of the regulatory requirement. Accurate assessment, however, is difficult because islets are spheroid-like cell aggregates of different sizes (<50 to 500 µm) resulting in possible thousandfold differences between the mass contribution of individual particles. The current standard manual counting method that uses size-based group classification is known to be error prone and operator dependent. Digital image analysis (DIA)-based methods can provide less subjective, more reproducible, and better-documented islet cell mass (IEQ) estimates; however, so far, none has become widely accepted or used. Here we present results obtained using a compact, self-contained islet cell counter (ICC3) that includes both the hardware and software needed for automated islet counting and requires minimal operator training and input; hence, it can be easily adapted at any center and could provide a convenient standardized cGMP-compliant IEQ assessment. Using cross-validated sample counting, we found that for most human islet cell preparations, ICC3 provides islet mass (IEQ) estimates that correlate well with those obtained by trained operators using the current manual SOP method ( r2 = 0.78, slope = 1.02). Variability and reproducibility are also improved compared to the manual method, and most of the remaining variability (CV = 8.9%) results from the rearrangement of the islet particles due to movement of the sample between counts. Characterization of the size distribution is also important, and the present digitally collected data allow more detailed analysis and coverage of a wider size range. We found again that for human islet cell preparations, a Weibull distribution function provides good description of the particle size.

Assessment of conjunctival goblet cell density using laser scanning confocal microscopy versus impression cytology.

PURPOSE: To determine the association between conjunctival goblet cell density (GCD) assessed using in vivo laser scanning confocal microscopy and conjunctival impression cytology in a healthy population. METHODS: Ninety (90) healthy participants undertook a validated 5-item dry eye questionnaire, non-invasive tear film break-up time measurement, ocular surface fluorescein staining and phenol red thread test. These tests where undertaken to diagnose and exclude participants with dry eye. The nasal bulbar conjunctiva was imaged using laser scanning confocal microscopy (LSCM). Conjunctival impression cytology (CIC) was performed in the same region a few minutes later. Conjunctival goblet cell density was calculated as cells/mm(2). RESULTS: There was a strong positive correlation of conjunctival GCD between LSCM and CIC (ρ=0.66). Conjunctival goblet cell density was 475±41 cells/mm(2) and 466±51 cells/mm(2) measured by LSCM and CIC, respectively. CONCLUSIONS: The strong association between in vivo and in vitro cellular analysis for measuring conjunctival GCD suggests that the more invasive CIC can be replaced by the less invasive LSCM in research and clinical practice.

A simple assessment model to quantifying the dynamic hippocampal neurogenic process in the adult mammalian brain.

Adult hippocampal neurogenesis is a highly dynamic process in which new cells are born, but only some of which survive. Of late it has become clear that these surviving newborn neurons have functional roles, most notably in certain forms of memory. Conventional methods to look at adult neurogenesis are based on the quantification of the number of newly born neurons using a simple cell counting methodology. However, this type of approach fails to capture the dynamic aspects of the neurogenic process, where neural proliferation, death and differentiation take place continuously and simultaneously. In this paper, we propose a simple mathematical approach to better understand the adult neurogenic process in the hippocampus which in turn will allow for a better analysis of this process in disease states and following drug therapies.

A study of cellular counting to determine minimum thresholds for adequacy for liquid-based cervical cytology using a survey and counting protocol.

BACKGROUND: Liquid-based cytology (LBC) for cervical screening would benefit from laboratory practice guidelines that define specimen adequacy for reporting of slides. The evidence base required to define cell adequacy should incorporate both ThinPrep™ (TP; Hologic, Inc., Bedford, MA, USA) and SurePath™ (SP; BD Diagnostics, Burlington, NC, USA), the two LBC systems used in the UK cervical screening programmes. OBJECTIVES: The objectives of this study were to determine (1) current practice for reporting LBC in England, Wales and Scotland, (2) a reproducible method for cell counting, (3) the cellularity of slides classified as inadequate, negative or abnormal and (4) the impact of varying cellularity on the likelihood of detecting cytological abnormalities. DESIGN: The study involved four separate arms to pursue each of the four objectives. (1) A questionnaire survey of laboratories was conducted. (2) A standard counting protocol was developed and used by three experienced cytopathologists to determine a reliable and reproducible cell counting method. (3) Slide sets which included a range of cytological abnormalities were each sent to three laboratories for cell counting to study the correlation between cell counts and reported cytological outcomes. (4) Dilution of LBC samples by fluid only (unmixed) or by dilution with a sample containing normal cells (mixed) was performed to study the impact on reporting of reducing either the total cell count or the relative proportion of abnormal to normal cells. SETTING: The study was conducted within the cervical screening programmes in England, Wales and Scotland, using routinely obtained cervical screening samples, and in 56 participating NHS cervical cytology laboratories. PARTICIPANTS: The study involved only routinely obtained cervical screening samples. INTERVENTIONS: There was no clinical intervention. MAIN OUTCOME MEASURES: The main outcome measures were (1) reliability of counting method, (2) correlation of reported cytology grades with cellularity and (3) levels of detection of abnormal cells in progressively diluted cervical samples. RESULTS: Laboratory practice varied in terms of threshold of cellular adequacy and of morphological markers of adequacy. While SP laboratories generally used a minimum acceptable cell count (MACC) of 15,000, the MACC employed by TP laboratories varied between 5000 and 15,000. The cell counting study showed that a standard protocol achieved moderate to strong inter-rater reproducibility. Analysis of slide reporting from laboratories revealed that a large proportion of the samples reported as inadequate had cell counts above a threshold of 15,000 for SP, and 5000 and 10,000 for TP. Inter-rater unanimity was greater among more cellular preparations. Dilution studies demonstrated greater detection of abnormalities in slides with counts above the MACC and among slides with more than 25 dyskaryotic cells. CONCLUSIONS: Variation in laboratory practice demonstrates a requirement for evidence-based standards for designating a MACC. This study has indicated that a MACC of 15,000 and 5000 for SP and TP, respectively, achieves a balance in terms of maintaining sensitivity and low inadequacy rates. FUTURE WORK: The findings of this study should inform the development of laboratory practice guidelines. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Trypanosoma cruzi infectivity assessment in "in vitro" culture systems by automated cell counting.

Chagas disease is an endemic, neglected tropical disease in Latin America that is caused by the protozoan parasite Trypanosoma cruzi. In vitro models constitute the first experimental approach to study the physiopathology of the disease and to assay potential new trypanocidal agents. Here, we report and describe clearly the use of commercial software (MATLAB(®)) to quantify T. cruzi amastigotes and infected mammalian cells (BeWo) and compared this analysis with the manual one. There was no statistically significant difference between the manual and the automatic quantification of the parasite; the two methods showed a correlation analysis r(2) value of 0.9159. The most significant advantage of the automatic quantification was the efficiency of the analysis. The drawback of this automated cell counting method was that some parasites were assigned to the wrong BeWo cell, however this data did not exceed 5% when adequate experimental conditions were chosen. We conclude that this quantification method constitutes an excellent tool for evaluating the parasite load in cells and therefore constitutes an easy and reliable ways to study parasite infectivity.

Compatibility of stabilized whole blood products with CD4 technologies and their suitability for quality assessment programs.

BACKGROUND: CD4 T cell enumeration is the most widely used prognostic marker for management of HIV disease. Internal quality control and external quality assessment (EQA) programs are critical to ensure reliability of clinical measurements. The utility of stabilized whole blood products (SWBP) as a test reagent for EQA programs such as Quality Assessment and Standardization for Immunological measures relevant to HIV/AIDS (QASI) program have been demonstrated previously. Since then, several new commercial SWBPs and alternative CD4 enumeration technologies have become available. Seven SWBPs were evaluated on seven different enumeration platforms to determine which product(s) are most suitable for EQA programs that support multiple analytical technologies. METHOD: Assessment of SWBPs was based on two criteria: (1) accuracy of CD4 T cell measurements and; (2) stability under sub optimal storage conditions. RESULTS: Three SWBPs (Multi-Check, StatusFlow and CD4 Count) showed accurate CD4 T-cell absolute count and percentage values across six of the enumeration platforms. All products retain stability up to 18 days at 21-23°C with the exception of Multi-Check-high on FacsCount and Multi-Check-Low and StatusFlow-Low on Pima. One of the products (CD4 Count) retained stability for three days on all platforms tested when stored at 37°C. CONCLUSION: This study demonstrated that the characteristics of commercially available SWBPs vary across multiple CD4 platforms. The compatibility of testing panels for EQA programs with multiple analytical platforms needs to be carefully considered, especially in large multiplatform CD4 EQA programs. The selection of a suitable cross-platform SWBP is an increasing challenge as more reagents and platforms are introduced for CD4 T-cell enumeration.

Automated assessment of ß-cell area and density per islet and patient using TMEM27 and BACE2 immunofluorescence staining in human pancreatic ß-cells.

In this study we aimed to establish an unbiased automatic quantification pipeline to assess islet specific features such as ß-cell area and density per islet based on immunofluorescence stainings. To determine these parameters, the in vivo protein expression levels of TMEM27 and BACE2 in pancreatic islets of 32 patients with type 2 diabetes (T2D) and in 28 non-diabetic individuals (ND) were used as input for the automated pipeline. The output of the automated pipeline was first compared to a previously developed manual area scoring system which takes into account the intensity of the staining as well as the percentage of cells which are stained within an islet. The median TMEM27 and BACE2 area scores of all islets investigated per patient correlated significantly with the manual scoring and with the median area score of insulin. Furthermore, the median area scores of TMEM27, BACE2 and insulin calculated from all T2D were significantly lower compared to the one of all ND. TMEM27, BACE2, and insulin area scores correlated as well in each individual tissue specimen. Moreover, islet size determined by costaining of glucagon and either TMEM27 or BACE2 and ß-cell density based either on TMEM27 or BACE2 positive cells correlated significantly. Finally, the TMEM27 area score showed a positive correlation with BMI in ND and an inverse pattern in T2D. In summary, automated quantification outperforms manual scoring by reducing time and individual bias. The simultaneous changes of TMEM27, BACE2, and insulin in the majority of the ß-cells suggest that these proteins reflect the total number of functional insulin producing ß-cells. Additionally, ß-cell subpopulations may be identified which are positive for TMEM27, BACE2 or insulin only. Thus, the cumulative assessment of all three markers may provide further information about the real ß-cell number per islet.

Primer evaluation and adaption for cost-efficient SYBR Green-based qPCR and its applicability for specific quantification of methanogens.

In the present study nine promising primer sets, targeting Archaea and methanogenic Archaea in particular, were evaluated in silico, in vitro and in situ concerning specificity, accuracy and applicability in end-point (ep-) and especially quantitative (q-)PCR research. The main goal was to adapt and evaluate already adapted primer sets, which were partially designed in combination with TaqMan probes, in substantially cheaper SYBR Green-based qPCR applications. An initial 16S rRNA gene bank-based in silico evaluation revealed high coverage potentials for all primers within targeted groups, ranging from 71 to 90%, except the Methanosaeta specific set showing a low potential of 37%. Mentionable cross-reacting potentials could be detected for the Methanothermobacter, Methanomicrobiales and Methanoculleus sets. The in vitro evaluation with selected reference organisms revealed a specific behavior for most primer sets, while the Methanosarcina and Methanothermobacter sets showed most problematic cross-reactions in epPCR application. We were able to show that primers for detecting the total archaeal community, methanogenic orders Methanosarcinales, Methanobacteriales, Methanococcales and the genus Methanoculleus performed in a highly specific way and allowed an accurate quantification of targeted organisms without the use of expensive TaqMan probes. However, primer pairs designed for detecting Methanomicrobiales, Methanothermobacter, Methanosarcina and Methanosaeta are not suitable for SYBR Green applications. The reliability of in situ quantifications was assessed for a typical methanogenic community, derived from a thermophilic fermenter, and confirmed via denaturing gradient gel band quantification and sequencing. Thereby, we revealed high abundances of methanogenic Archaea, mainly comprising Methanoculleus and Methanosarcinales, while Methanobacteriales only formed a minor fraction.

Assessment of goblet cell orifice distribution across the rabbit bulbar conjunctiva based on numerical density and nearest neighbors analysis.

PURPOSE: To assess density and spatial distribution of the goblet cell orifices at the surface of the rabbit bulbar conjunctiva as an indicator of functional activity. METHODS: Specimens of the superior or inferior bulbar conjunctiva from six healthy young adult (2 kg) pigmented rabbits were obtained using a special preparation technique by which the conjunctiva was carefully stretched out during fixation with buffered glutaraldehyde. The apical surface of the specimens was examined by scanning electron microscopy. From high magnification prints, the areas and dimensions of 32-49 orifices/image were measured. In addition, the centre-to-centre spacing and spatial distribution of the orifices were assessed using a nearest neighbors principle. RESULTS: The bulbar conjunctival surface is composed of polygonal cells decorated with surface microplicae and in between which are individual goblet cell orifices. The goblet cell orifices are characterized by tending to be oval in shape (long:short dimensions ratio of 1.57 +/- 0.23) and usually having a distinct line of microvilli around the perimeter. The orifices had a wide range of areas (from 13 to 188 µm(2); group mean +/- SD of 54 +/- 36 µm(2)), and distribution of orifice areas was skewed or even bimodal. The overall orifice density was 387 +/- 68/mm(2), with the group-averaged nearest neighbors distance being 34 +/- 3 µm. Comparisons of the measured nearest neighbors distances to that for an optimum spacing based on numerical density reveals the goblet cell orifices to be slightly further apart and that they were not obviously in groups or clustered. CONCLUSIONS: Goblet cell orifices at the bulbar conjunctival surface, a presumed indicator of functional secretory activity, appear to have reproducible density and a discrete and reasonably predictable spatial distribution.