Association Between the Severity of Diabetic Retinopathy and Optical Coherence Tomography Angiography Metrics.

Diabetic retinopathy, the most serious ocular complication of diabetes, imposes a serious economic burden on society. Automatic and objective assessment of vessel changes can effectively manage diabetic retinopathy and prevent blindness. Optical coherence tomography angiography (OCTA) metrics have been confirmed to be used to assess vessel changes. The accuracy and reliability of OCTA metrics are restricted by vessel segmentation methods. In this study, a multi-branch retinal vessel segmentation method is proposed, which is comparable to the segmentation results obtained from the manual segmentation, effectively extracting vessels in low contrast areas and improving the integrity of the extracted vessels. OCTA metrics based on the proposed segmentation method were validated to be reliable for further analysis of the relationship between OCTA metrics and diabetes and the severity of diabetic retinopathy. Changes in vessel morphology are influenced by systemic risk factors. However, there is a lack of analysis of the relationship between OCTA metrics and systemic risk factors. We conducted a cross-sectional study that included 362 eyes of 221 diabetic patients and 1,151 eyes of 587 healthy people. Eight systemic risk factors were confirmed to be closely related to diabetes. After controlling these systemic risk factors, significant OCTA metrics (such as vessel complexity index, vessel diameter index, and mean thickness of retinal nerve fiber layer centered in the macular) were found to be related to diabetic retinopathy and severe diabetic retinopathy. This study provides evidence to support the potential value of OCTA metrics as biomarkers of diabetic retinopathy.

An evaluation of existing manual blood film schistocyte quantitation guidelines and a new proposed method.

Schistocytosis is the morphological hallmark of the microangiopathic haemolytic anaemia of thrombotic microangiopathy (TMA). Consensus guidelines for manual schistocyte quantitation are available, but limited research has evaluated them. The 2012 International Council for Standardization in Haematology (ICSH) recommends a schistocyte quantitation of 1% as a robust cut-off for significance, with the quantitation including helmet, crescent, triangle and keratocyte poikilocytes; and microspherocytes only in the presence of helmets, crescents/triangles, and keratocytes. We aimed to evaluate the relative contribution of these different poikilocytes to schistocyte counting; compare the ICSH method with our proposed method which counts only cells most specific for red cell fragmentation (helmet, crescent and triangular schistocytes); and evaluate inter- and intra-observer agreement. Blood films were sourced from the Australian Snakebite Project, including non-envenomed and envenomed cases, with and without TMA. In blood films across the range of schistocytosis, the predominant poikilocytes present were helmets and crescents. Triangles, keratocytes and microspherocytes were typically only present when ICSH schistocyte count was >1%. With results dichotomised as <1.0% or ≥1.0%, our proposed new method versus the ICSH method showed almost perfect agreement [observed agreement 95%, Cohen's kappa (κ)=0.84, SE 0.04, 95% CI 0.76-0.92, p<0.005]. Inter-observer strength of agreement for our method was moderate (Fleiss' κ for comparisons between three non-unique microscopists κ=0.50, SE 0.05, 95% CI 0.41-0.59, p<0.005). Intra-observer reproducibility assessed in two microscopists ranged from substantial (Cohen's κ=0.71, SE 0.08, 95% CI 0.55-0.86, p<0.005) to borderline almost perfect agreement (Cohen's κ=0.81, SE 0.07, 95% CI 0.68-0.93, p<0.005). Schistocyte quantitation using our new method is simpler than the 2012 ICSH method and had almost perfect agreement. Our finding of moderate inter-observer agreement in quantitating helmet, triangle and crescent schistocytes is applicable to both the ICSH and our newly proposed method. This finding underscores the importance of clinicopathological correlation and repeated examinations in the context of a clinically suspected TMA.

Myopia, corneal endothelial cell density and morphology in a Japanese population-based cross-sectional study: the JPHC-NEXT Eye Study.

This population-based cross-sectional study was performed to determine the mean corneal endothelial cell density (ECD), coefficient of variation (CV), and hexagonality (HEX), and their associations with myopia in Japanese adults living in Chikusei city. Of 7109 participants with available data, 5713 (2331 male and 3382 female) participants were eligible for analysis. After assessing the relationship between participant characteristics and spherical equivalent refraction (SER), the association of SER with the abnormal value of ECD (< 2000 cells/mm), CV (≥ 0.40), and HEX (≤ 50%) were determined using the logistic regression models adjusting for potential confounders (age, intraocular pressure, keratometric power, height, and antihypertensive drug use). In male participants, there was no statistically significant relationships between SER and endothelial parameters. In female participants, compared to emmetropia, SER ≤ - 6 D had significantly higher odds ratio (OR) of having the abnormal value of CV (OR = 2.07, 95% confidence interval [CI] 1.39-3.10) and HEX (OR = 2.04, 95% CI 1.29-3.23), adjusted for potential confounders, indicating that the high myopia was associated with the abnormal values of CV and HEX. Further adjustment for contact lenses wear partly attenuated these associations. Association between the SER and ECD was not detected.

Prognostic Threshold for Circulating Tumor Cells in Patients With Pancreatic and Midgut Neuroendocrine Tumors.

BACKGROUND: Circulating tumor cells (CTCs) are detectable in patients with neuroendocrine tumors (NETs) and are accurate prognostic markers although the optimum threshold has not been defined. OBJECTIVE: This work aims to define optimal prognostic CTC thresholds in PanNET and midgut NETs. PATIENTS AND METHODS: CellSearch was used to enumerate CTCs in 199 patients with metastatic pancreatic (PanNET) (90) or midgut NETs (109). Patients were followed for progression-free survival (PFS) and overall survival (OS) for a minimum of 3 years or until death. RESULTS: The area under the receiver operating characteristic curve (AUROC) for progression at 12 months in PanNETs and midgut NETs identified the optimal CTC threshold as 1 or greater and 2 or greater, respectively. In multivariate logistic regression analysis, these thresholds were predictive for 12-month progression with an odds ratio (OR) of 6.69 (P < .01) for PanNETs and 5.88 (P < .003) for midgut NETs. The same thresholds were found to be optimal for predicting death at 36 months, with an OR of 2.87 (P < .03) and 5.09 (P < .005) for PanNETs and midgut NETs, respectively. In multivariate Cox hazard regression analysis for PFS in PanNETs, 1 or greater CTC had a hazard ratio (HR) of 2.6 (P < .01), whereas 2 or greater CTCs had an HR of 2.25 (P < .01) in midgut NETs. In multivariate analysis OS in PanNETs, 1 or greater CTCs had an HR of 3.16 (P < .01) and in midgut NETs, 2 or greater CTCs had an HR of 1.73 (P < .06). CONCLUSIONS: The optimal CTC threshold to predict PFS and OS in metastatic PanNETs and midgut NETs is 1 and 2, respectively. These thresholds can be used to stratify patients in clinical practice and clinical trials.

Comparison of two methods for cell count determination in the course of biocide susceptibility testing.

The inoculum density is an important parameter for numerous experimental approaches in bacteriology, including antimicrobial susceptibility testing (AST), biocide susceptibility testing (BST) and biocide efficacy testing (BET). Methods to determine the inoculum density commonly refer to cell counts and have been described for BET according to the German Medical Veterinary Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) and for AST according to the Clinical and Laboratory Standards Institute (CLSI). In this study, the DVG method using 1000 µL volumes of two different dilution steps and the AST method according to CLSI using a 100 µL volume of a single dilution step from the inoculum suspension were compared. For this, each of the four reference strains, Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442, was comparatively tested 28 times using the inoculum preparation according to DVG. The results were statistically analysed using Bland-Altman plots and 95 % limits of agreement (AL). Moreover, cell counts were correlated with the optical density of the bacterial suspensions used. In comparison, the CLSI method measured lower values for colony-forming units (CFU) of -0.12 log10 compared to the DVG method. Overall, both methods returned an AL of -0.52 to 0.27 log10. Since the variations observed between the two methods were within one log10 step and the measured CFUs did not differ systematically, both methods proved to be suitable for cell count determination. Therefore, the CLSI method, which is less complex and less time-consuming, is recommended.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants.

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

Reevaluation of reference values for bone marrow differential counts in 236 healthy bone marrow donors.

Despite the increasing role of molecular markers, differential counts and morphology of hematopoietic cells in the bone marrow (BM) remain essential diagnostic criteria in hematological diseases. However, the respective reference values for BM myelogram commonly used came from small series with limited numbers of healthy individuals. We evaluated the myelograms of 236 healthy individuals who underwent unrelated bone marrow donation. Health check-ups were performed 4 weeks prior to harvest. Samples for this study, taken from the first aspiration, were stained according to the standard Pappenheim method. Three experienced investigators assessed cellularity, megakaryopoiesis, and differential counts independently. The median donor age was 31 (range 18-51) years. Predonation tests did not reveal any relevant morbidity. Thirty-seven out of 42 hypocellular marrow samples were from younger donors up to 39 years. Content of megakaryocytes was normal in 210 specimens (89%). Gender and body mass index had significant impact on hematopoiesis, whereas age had not. The number of erythroblasts was higher (about 32%) and the proportion granulopoiesis slightly lower (about 50%) compared with previous studies. Differential counts showed also some differences with respect to individual maturation stages in these lines. Interrater comparisons showed greater reliability for the assignment of cells to the different hematopoietic cell lines than for single-cell diagnoses. This study largely confirms the results for cell counts in normal human bone marrow available from previous reports and provides some insights into factors that affect individual cell populations. It also reveals substantial variability among even experienced investigators in cytological diagnoses.

Heterogeneity of Human Pancreatic Islet Isolation Around Europe: Results of a Survey Study.

BACKGROUND: Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries. METHODS: A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan. RESULTS: Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities. CONCLUSIONS: The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.

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.

Evaluation of the predictive value of the hematopoietic progenitor cell count using an automated hematology analyzer for CD34+ stem cell mobilization and apheresis product yield.

INTRODUCTION: We evaluated the value of hematopoietic progenitor cells (HPCs) counted in Sysmex XN analyzers to predict the mobilization and collection of CD34+ cells in apheresis for stem cell transplantation. METHODS: Eighty patients who underwent stem cell transplantation were enrolled (50 autologous and 30 allogeneic). In the autologous group, patients were considered poor mobilizers when the CD34+ count was <10 × 106 /L or <20 × 106 /L in patients with multiple myeloma who were going to undergo two transplants. ROC curves were generated, and HPC cutoffs were calculated. RESULTS: The correlation between the HPC and CD34+ cell counts was good. Two algorithms were proposed. In the first algorithm, samples collected the day before apheresis, negative and positive HPC cutoffs were selected to detect poor and good mobilization and, therefore, the need or not to administer plerixafor. In the second algorithm, samples collected pre-apheresis, the negative HPC cutoff was an indication to delay apheresis; an HPC higher than the optimal cutoff was an indication to start apheresis. When the HPC values were between these cutoffs, there was an indication to count CD34+ cells for a better decision-making. Finally, in samples collected pre-apheresis, HPC counts could be used to predict patients who would have poor CD34+ cell collections. In the allogeneic group, all the donors mobilized well, and very few needed two apheresis procedures. CONCLUSIONS: The HPC count is useful for decision-making in the management of patients subjected to apheresis procedures to collect peripheral blood stem cells.

Automated Counting of Cancer Cells by Ensembling Deep Features.

High-content and high-throughput digital microscopes have generated large image sets in biological experiments and clinical practice. Automatic image analysis techniques, such as cell counting, are in high demand. Here, cell counting was treated as a regression problem using image features (phenotypes) extracted by deep learning models. Three deep convolutional neural network models were developed to regress image features to their cell counts in an end-to-end way. Theoretically, ensembling imaging phenotypes should have better representative ability than a single type of imaging phenotype. We implemented this idea by integrating two types of imaging phenotypes (dot density map and foreground mask) extracted by two autoencoders and regressing the ensembled imaging phenotypes to cell counts afterwards. Two publicly available datasets with synthetic microscopic images were used to train and test the proposed models. Root mean square error, mean absolute error, mean absolute percent error, and Pearson correlation were applied to evaluate the models' performance. The well-trained models were also applied to predict the cancer cell counts of real microscopic images acquired in a biological experiment to evaluate the roles of two colorectal-cancer-related genes. The proposed model by ensembling deep imaging features showed better performance in terms of smaller errors and larger correlations than those based on a single type of imaging feature. Overall, all models' predictions showed a high correlation with the true cell counts. The ensembling-based model integrated high-level imaging phenotypes to improve the estimation of cell counts from high-content and high-throughput microscopic images.

The reliability of the isotropic fractionator method for counting total cells and neurons.

BACKGROUND: The Isotropic Fractionator (IF) is a method to determine the cellular composition of nervous tissue. It has been mostly applied to assess variation across species, where differences are expected to be large enough not to be masked by methodological error. However, understanding the sources of variation in the method is important if the goal is to detect smaller differences, for example, in same-species comparisons. Comparisons between different mice strains suggest that the IF is consistent enough to detect these differences. Nevertheless, the reliability of the method has not yet been examined directly. METHOD: In this study, we evaluate the reliability of the method for the determination of cellular and neuronal numbers of Swiss mice. We performed repeated cell counts of the same material by different experimenters to quantify different sources of variation. RESULTS: In total cell counts, we observed that for the cerebral cortex most of the variance was at the counter level. For the cerebellum, most of the variance is attributed to the sample itself. As for neurons, random error along with the immunostaining correspond to most of the variation, both in the cerebral cortex and in the cerebellum. Test-retest reliability coefficients were relatively high, especially for cell counts. CONCLUSIONS: Although biases between counters and random variation in staining could be problematic when aggregating data from different sources, we offer practical suggestions to improve the reliability of the method. While small, this study is a most needed step towards more precise measurement of the brain's cellular composition.

Accurate and reproducible enumeration of T-, B-, and NK lymphocytes using the BD FACSLyric 10-color system: A multisite clinical evaluation.

Clinical flow cytometry is a reliable methodology for whole blood cell phenotyping for different applications. The BD FACSLyric™ system comprises a flow cytometer available in different optical configurations, BD FACSuite™ Clinical software, and optional BD FACS™ Universal Loader. BD FACSuite Clinical software used with BD™ FC Beads and BD CS&T Beads enable universal setup for performance QC, instrument control, data acquisition/storage, online/offline data analysis, and instrument standardization. BD Biosciences sponsored the clinical evaluation of the BD FACSLyric 10-color configuration at seven clinical sites using delinked and de-identified blood specimens from HIV-infected and uninfected subjects to enumerate T-, B-, and NK-lymphocytes with the BD Multitest™ reagents (BD Multitest IMK kit and BD Multitest 6-color TBNK). Samples were analyzed on the BD FACSLyric system with BD FACSuite Clinical software, and on the BD FACSCanto™ II system with BD FACSCanto clinical software and BD FACS 7-Color Setup beads. For equivalency between methods, data (n = 362) were analyzed with Deming regression for absolute count and percentage of lymphocytes. Results gave R2 ≥0.98, with slope values ≥0.96, and slope ranges between 0.90-1.05. The percent (%) bias values were <10% for T- and NK cells and <15% for B- cells. The between-site (n = 4) total precision was tested for 5 days (2 runs/day), and gave %coefficient of variation below 10% for absolute cell counts. The stability claims were confirmed (n = 186) for the two BD Multitest reagents. The reference intervals were re-established in male and female adults (n = 134). The analysis by gender showed statistically significant differences for CD3+ and CD4+ T-cell counts and %CD4. In summary, the BD FACSLyric and the BD FACSCanto II systems generated comparable measurements of T-, B-, and NK-cells using BD Multitest assays.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method.

We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Evaluation of the effect of storage condition on cell extraction and flow cytometric analysis from intestinal biopsies.

BACKGROUND: Flow cytometric (FC) analysis of intestinal tissue biopsies requires prompt cell isolation and processing to prevent cell death and generate valid data. We examined the effect of storage conditions prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies. METHODS: Biopsies (N = 224) from inflamed or non-inflamed ileal and/or colonic tissue from three patients with Crohn's disease were processed and analyzed immediately in duplicate, or stored under different conditions. Cells were isolated and stained for specific markers, followed by FC. RESULTS: Decreased mean live CD45+ cell counts were observed after storage of biopsies at -80 °C dimethyl sulfoxide (DMSO)/citrate buffer compared with immediate processing (1794.3 vs. 19,672.7; p = 0.006]). A non-significant decrease in CD45+ live cell count occurred after storage at -20 °C in DMSO/citrate buffer and cell yield was adequate for subsequent analysis. CD3+ cell proportions were significantly lower after storage at 4 °C in complete medium for 48 h compared with immediate analysis. Mean CD14+ cell proportions were significantly higher after storage of biopsies at -80 °C in DMSO/citrate buffer compared with immediate analysis (2.61% vs. 1.31%, p = 0.007). CD4+, CD8+ and CD4+/CD8+ cell proportions were unaffected by storage condition. CONCLUSION: Storage of intestinal tissue biopsies at -20 °C in DMSO/citrate buffer for up to 48 h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. These findings support the potential shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials.

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.

In search for a gold-standard procedure to count motor neurons in the spinal cord.

Counting motor neurons within the spinal cord and brainstem represents a seminal step to comprehend the anatomy and physiology of the final common pathway sourcing from the CNS. Motor neuron loss allows to assess the severity of motor neuron disorders while providing a tool to assess disease modifying effects. Counting motor neurons at first implies gold standard identification methods. In fact, motor neurons may occur within mixed nuclei housing a considerable amount of neurons other than motor neurons. In the present review, we analyse various approaches to count motor neurons emphasizing both the benefits and bias of each protocol. A special emphasis is placed on discussing automated stereology. When automated stereology does not take into account site-specificity and does not distinguish between heterogeneous neuronal populations, it may confound data making such a procedure a sort of "guide for the perplex". Thus, if on the one hand automated stereology improves our ability to quantify neuronal populations, it may also hide false positives/negatives in neuronal counts. For instance, classic staining for antigens such as SMI-32, SMN and ChAT, which are routinely considered to be specific for motor neurons, may also occur in other neuronal types of the spinal cord. Even site specificity within Lamina IX may be misleading due to neuronal populations having a size and shape typical of motor neurons. This is the case of spinal border cells, which often surpass the border of Lamina VII and intermingle with motor neurons of Lamina IX. The present article discusses the need to join automated stereology with a dedicated knowledge of each specific neuroanatomical setting.

Effects of diluents on cell culture viability measured by automated cell counter.

Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate inherent variability in subjective interpretation associated with manual hemocytometers. When using these cell counters, sample dilution is often necessary to stay within the assay measurement range; however, the effect of time and diluents on cell culture is not well understood. This report presents the adverse effect of phosphate buffered saline as a diluent on cell viability when used in combination with an automated cell counter. The reduced cell viability was attributed to shear stress introduced by the automated cell counter. Furthermore, length of time samples were incubated in phosphate buffered saline also contributed to the observed drop in cell viability. Finally, as erroneous viability measurements can severely impact process decisions and product quality, this report identifies several alternative diluents that can maintain cell culture viability over time in order to ensure accurate representation of cell culture conditions.