Evaluating The Quality Of Cell Counting Measurements Using Experimental Design And Statistical Analysis

Cell counting measurements are critical in the research, development, and manufacturing of cell therapy products where they support decision-making in product testing and release. Evaluating cell quantity with accuracy and precision has remained a challenge for many specific applications or purposes. While new measurement platforms have been developed with increased measurement throughput and improved precision, discrepancies between cell counts acquired via various measurement processes are still pervasive. In addition, the industry as a whole has recognized that complex biological properties, as well as operator, equipment, and procedure variations can greatly affect the measurement quality, and the development of a single reference material or reference measurement is impractical to address broad counting needs. Here, we describe an experimental design and statistical analysis approach to evaluate the quality of a given cell counting measurement process. The experimental design uses a dilution series study with replicate samples as well as procedures to reduce pipetting error, and operator and temporal bias. The statistical analysis methods generate a set of metrics for evaluating measurement quality in terms of accuracy and precision, where accuracy is based on deviation from proportionality. In this design, a proportional response to dilution fraction serves as an internal control, where deviation from a proportional response is indicative of a systematic or non-systematic bias in the measurement process. The utility of this approach was demonstrated in the counting of human mesenchymal stem cells (hMSC) via automated or manual counting methods, where the automated method performed better in terms of both precision and proportionality. These results enabled a transition from the labor intensive and often imprecise manual counting method to the automated counting method. The experimental design and statistical method presented here is agnostic to the cell type and analytical platform, thus suitable as a horizontal approach to evaluate the quality of cell counting measurements with respect to method selection, optimization, and validation, thereby facilitating subsequent decision making. We are also working closely with industry partners and Standards Development Organizations (SDOs) to develop cell counting standards using this and other strategies

Thin films of Type 1 collagen for cell by cell analysis of morphology and tenascin-C promoter activity

BACKGROUND: The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of cells on different extracellular matrices. We have quantitatively examined the relationship between fibroblast morphology and activation of the promoter for the extracellular matrix protein tenascin-C using a tenascin-C promoter-based GFP reporter construct. RESULTS: We find that when considering the average response from the population of cells, cell area correlates with tenascin-C promoter activity as has been previously suggested; however cell-by-cell analysis suggests that cell area and promoter activity are not tightly correlated within individual cells. CONCLUSION: This study demonstrates how quantitative cell-by-cell analysis, facilitated by the use of thin films of extracellular matrix proteins, can provide insight into the relationship between phenotypic parameters

Piping plovers demonstrate regional differences in nesting habitat selection patterns along the U. S. Atlantic coast

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Zeigler, S. L., Gutierrez, B. T., Hecht, A., Plant, N. G., & Sturdivant, E. J. Piping plovers demonstrate regional differences in nesting habitat selection patterns along the U. S. Atlantic coast. Ecosphere, 12(3), (2021): e03418, https://doi.org/10.1002/ecs2.3418.Habitat studies that encompass a large portion of a species’ geographic distribution can explain characteristics that are either consistent or variable, further informing inference from more localized studies and improving management successes throughout the range. We identified landscape characteristics at Piping Plover nests at 21 sites distributed from Massachusetts to North Carolina and compared habitat selection patterns among the three designated U.S. recovery units (New England, New York–New Jersey, and Southern). Geomorphic setting, substrate type, and vegetation type and density were determined in situ at 928 Piping Plover nests (hereafter, used resource units) and 641 random points (available resource units). Elevation, beach width, Euclidean distance to ocean shoreline, and least-cost path distance to low-energy shorelines with moist substrates (commonly used as foraging habitat) were associated with used and available resource units using remotely sensed spatial data. We evaluated multivariate differences in habitat selection patterns by comparing recovery unit-specific Bayesian networks. We then further explored individual variables that drove disparities among Bayesian networks using resource selection ratios for categorical variables and Welch’s unequal variances t-tests for continuous variables. We found that relationships among variables and their connections to habitat selection were similar among recovery units, as seen in commonalities in Bayesian network structures. Furthermore, nesting Piping Plovers consistently selected mixed sand and shell, gravel, or cobble substrates as well as areas with sparse or no vegetation, irrespective of recovery unit. However, we observed significant differences among recovery units in the elevations, distances to ocean, and distances to low-energy shorelines of used resource units. Birds also exhibited increased selectivity for overwash habitats and for areas with access to low-energy shorelines along a latitudinal gradient from north to south. These results have important implications for conservation and management, including assessment of shoreline stabilization and habitat restoration planning as well as forecasting effects of climate change.Funding for this work was provided by the North Atlantic Landscape Conservation Cooperative and U.S. Fish and Wildlife Service through a U.S. Geological Survey Mendenhall Fellowship to Zeigler. All other funding was through the U.S. Geological Survey (Zeigler, Gutierrez, Plant, and Sturdivant) and the U.S. Fish and Wildlife Service (Hecht). Zeigler, Plant, and Hecht conceived and designed the study and secured funding

Long-term stability of anti-cyclic citrullinated peptide antibody status in patients with early inflammatory polyarthritis.

INTRODUCTION: The utility of reassessing anti-cyclic citrullinated peptide (anti-CCP) antibody status later in disease in patients presenting with early undifferentiated inflammatory polyarthritis, particularly in those who test negative for both anti-CCP and rheumatoid factor (RF) at baseline, remains unclear. We aimed therefore to determine the stability of CCP antibody status over time and the prognostic utility of repeated testing in subjects with early inflammatory polyarthritis (IP). METHODS: Anti-CCP and RF were measured at baseline and 5 years in 640 IP patients from the Norfolk Arthritis Register, a primary care-based inception cohort. The relation between change in anti-CCP status/titer and the presence of radiologic erosions, the extent of the Larsen score, and Health Assessment Questionnaire (HAQ) score by 5 years was investigated. RESULTS: With a cut-off of 5 U/ml, 28% subjects tested positive for anti-CCP antibodies, 29% for RF, and 21% for both at baseline. Nine (2%) anti-CCP-negative patients seroconverted to positive, and nine (4.6%) anti-CCP-positive individuals became negative between baseline and 5 years. In contrast, RF status changed in 17% of subjects. However, change in RF status was strongly linked to baseline anti-CCP status and was not independently associated with outcome. Ever positivity for anti-CCP antibodies by 5 years did not improve prediction of radiographic damage compared with baseline status alone (accuracy, 75% versus 74%). A higher baseline anti-CCP titer (but not change in anti-CCP titer) predicted worse radiologic damage at 5 years (P < 0.0001), even at levels below the cut-off for anti-CCP positivity. Thus, a titer of 2 to 5 U/ml was strongly associated with erosions by 5 years (odds ratio, 3.6 (1.5 to 8.3); P = 0.003). CONCLUSIONS: Repeated testing of anti-CCP antibodies or RF in patients with IP does not improve prognostic value and should not be recommended in routine clinical practice.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Chromatographic Separation Apparatus

An apparatus for the monitoring of a column chromatography separation process includes a segmented column with a seal positioned at the joint defined by the segments of the column. A connector is provided for connecting the segments of the column together. The apparatus further includes a sensor for monitoring an analyte in an eluant within a separation zone of the column. The sensor includes a mesh grid made of optical fibers or metal wires which is placed so as to extend through the separation zone of the column. The metal wires or optical fibers extend through the seal of the joint in the segmented column and connect to signal processing and data analysis equipment for purposes of monitoring the movements and concentration of an analyte in an eluant at various locations within the column. Certain segments of the optical fibers or metal wires which make up the mesh grid are coated so as to be desensitized and other segments are uncoated for sensing the analyte. This provides an effective apparatus to monitor in detail the cross-section of a column chromatography process in-situ

Radiologic imaging in cystic fibrosis: cumulative effective dose and changing trends over 2 decades

Objective: With the increasing life expectancy for patients with cystic fibrosis (CF), and a known predisposition to certain cancers, cumulative radiation exposure from radiologic imaging is of increasing significance. This study explores the estimated cumulative effective radiation dose over a 17-year period from radiologic procedures and changing trends of imaging modalities over this period. Methods: Estimated cumulative effective dose (CED) from all thoracic and extrathoracic imaging modalities and interventional radiology procedures for both adult and pediatric patients with CF, exclusively attending a nationally designated CF center between 1992-2009 for > 1 year, was determined. The study period was divided into three equal tertiles, and estimated CED attributable to all radiologic procedures was estimated for each tertile. Results: Two hundred thirty patients met inclusion criteria (2,240 person-years of follow-up; 5,596 radiologic procedures). CED was > 75 mSv for one patient (0.43%), 36 patients (15.6%) had a CED between 20 and 75 mSv, 56 patients (24.3%) had a CED between 5 and 20 mSv, and in 138 patients (60%) the CED was estimated to be between 0 and 5 mSv over the study period. The mean annual CED per patient increased consecutively from 0.39 mSv/y to 0.47 mSv/y to 1.67 mSv/y over the tertiles one to three of the study period, respectively (P < .001). Thoracic imaging accounted for 46.9% of the total CED and abdominopelvic imaging accounted for 42.9% of the CED, respectively. There was an associated 5.9-fold increase in the use of all CT scanning per patient (P < .001). Conclusions: This study highlights the increasing exposure to ionizing radiation to patients with CF as a result of diagnostic imaging, primarily attributable to CT scanning. Increased awareness of CED and strategies to reduce this exposure are needed