Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.     

The routine differential leukocyte count vs automated differential counts

The clinical use of the proposed performance standards for differential leukocyte counts is determined by multiple factors. Their use must be considered according to the specific use of the differential count, the sources of variability in differential counting, the relation between specific use and sources of variability, the role of analytic errors in the detection of nonspecific changes, the use of qualitative vs quantitative data, the sensitivity and specificity of the routine eye count, the role of disease or specific cell prevalence in determination of predictive value, the effect on use of automated instruments for screening, and whether abnormal specimen flagging can be done. The routine eye-count differential method, as performed by a well-trained technologist or technician, seems to lack both sensitivity and specificity. Because of the magnitude of technique- and method-related and biologic sources of variability, the 100- or 200-cell eye-count differential method is not a good screening method for detection of hematologic illnesses, particularly those that are uncommon. The automated differential leukocyte instruments address many of the technique- and method-related errors and are thus able to equal or exceed the performance of the routine eye-count differential method.     

Comparison of methods for assessing integrity of equine sperm membranes

Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = −3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = −11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.     

Immune modulation in the guinea pig using cortisone acetate.

Cortisone acetate was administered to a group of guinea pigs (Cavia porcellus) at 0 (control), 20 (low) or 200 (high) mg/kg. Steroid was given daily for two individual 7 day periods, separated by 7 days of no treatment. The effects of this steroid on body weight gain, thymic weight, total and differential leukocyte counts, serum antibody titer against a bacterin, dermal hypersensitivity response to a sensitizing agent and histological evaluation of lymphoid and other tissues were evaluated. Significant differences in body weight gain (p less than 0.05) and thymic weight (p less than .01) were noted. For total leukocyte count, no significant difference among treatment groups at individual time points was noted (p greater than .10), while significant differences were seen in lymphocyte and neutrophil counts (p less than .01). A significant difference in antibody titer among the treatment groups was observed (p less than .01). For the dermal hypersensitivity response, there was no consistent pattern among the treatment groups in gross (macroscopic) skin reactions. Microscopically, differences were seen in the inflammatory response among the treatment groups. Histologically, steroid related changes were seen in thymus, spleen, lymph node and liver. At necropsy, 24 of 40 animals had lesions of focal necrotizing hepatitis. Three affected animals died and remaining animals showed no clinical illness. The cause of the necrotizing hepatitis could not be determined by culture, special stains, electron microscopy, serology or by attempts at transmission with affected liver samples.     

Accuracy of routine flow-cytometric bitmap selection for three leukocyte populations

A double-blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts. White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS-C flow cytometer using the two-parameter 90 degrees light scatter vs. forward angle light scatter (granularity vs. cell size) data distribution. Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each. The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer. Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps. Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps. Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential. In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.     

Automatic enumeration of adherent streptococci or actinomyces on dental alloy by fluorescence image analysis.

The aim of the present study was to develop an automated image analysis method to quantify adherence of Streptococcus sanguinis or Actinomyces viscosus on surfaces of a currently used dental alloy. Counting such bacterial strains was difficult because of their arrangement, thus S. sanguinis being a coccus arranged in chains or pairs, and A. viscosus a long complexly arranged polymorph rod. Direct counting of fluorescently stained adherent bacteria was done visually and with image analysis methods. To differentiate these two morphotypes, two programs were developed: (i) for streptococci, thresholding and selection of the object maxima, and (ii) for actinomyces, two step thresholding and processing of the characteristic points of the object skeletons. The triplicate enumerations for each bacterial strain were not significantly different (p > 0.005) and correlations between visual counting and automated counting were significant (r = 0.91 for S. sanguinis and r = 0.99 for A. viscosus, p <00.0001). These rapid and reproducible methods, allowed us to count either cocci or rods, adherent on an inert substratum, in high density conditions.     

Application of a non-hazardous vital dye for cell counting with automated cell counters

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.     

Hematology of sloth bears (Melursus ursinus ursinus) from two locations in India

Standard hematology parameters were determined for 122 sloth bears (Melursus ursinus ursinus) at the Sur Sarovar Bird Sanctuary, Uttar Pradesh, India (27 degrees 0'N; 77 degrees 45'E), and the Bannerghatta Biological Park, Karnataka, India (12 degrees 48'N; 77 degrees 34'E) from March 2003 to July 2006. These two native sloth bear habitats have different climatic conditions and provided an opportunity to examine the effect of climate on the physiologic hematology values of these bears. We primarily analyzed the influence of age, sex, season, and body weight on the different hematology parameters. Several values were significantly different in sloth bear cubs (1 yr). The cubs had a lower erythrocyte count, hemoglobin concentration, packed cell volume (PCV), and mean cell hemoglobin (MCV) values when compared to adult and subadult bears. The cubs also had higher leukocyte counts, due to higher circulating neutrophils, as compared to adult and subadult bears. Within subadult and adult bears, we also identified a sexual dimorphic difference in leukocyte count in adult and subadult bears, wherein female bears had higher counts than males. This difference was the result of a significantly higher number of circulating neutrophils in female bears. Platelet counts were also higher in females as compared to males. On comparing different seasons, leukocyte counts were higher in winter as compared to the summer and monsoon seasons. When compared based on location, erythrocyte counts were higher in subadult and adult bears at Bannerghatta, which was at a higher altitude than Sur Sarovar. Within subadult and adult bears, we did not find any significant influence of age or body weight on the different hematologic parameters. In this study we have obtained mean hematologic values for sloth bears in their native habitat to serve as a reference for this species. This report will be useful to develop and evaluate health profiles of sloth bears under various ecological conditions.     

Imprecision of ratio-derived differential leukocyte counts

Differential leukocyte counts are very imprecise if determined as the product of a total WBC and a percentage obtained by differentiation of a relatively small number of cells. The imprecision can be decreased by increasing simultaneously the numbers of both the counted and the differentiated cells. An increase of only one (such as by automated counting of the WBC) without a simultaneous increase of the other one does not lead to an appreciable reduction of the imprecision.     

Comparison of performance for leukocyte differential counting of the Technicon H6000 system with a manual reference method using the NCCLS standard

The National Committee for Clinical Laboratory Standards (NCCLS) has published a tentative standard for leukocyte differential counting, by means of which a manual or automated method for leukocyte differential counting can be compared with a manual reference method. The performance of the Technicon H6000 system was evaluated using the standard at Stamford and Overlook Hospitals. A total of 502 patient samples were analyzed: 315 from Overlook and 187 from Stamford. The H6000 system was found to be approximately four times more precise than the 200-cell manual reference method for each cell type. Correlation of the H6000 system with the manual method was good, with correlation coefficients of 0.98 for neutrophils and lymphocytes, 0.96 for eosinophils, 0.72 for monocytes, and 0.5 for basophils. The clinical sensitivity of the H6000 system, measured in terms of false normals and false abnormals, was similar to that of the manual reference method when measured against itself. There were no clinically significant discrepancies in results from the H6000 system, except for possibly one case where a patient was already on antibiotic therapy. The NCCLS standard was found to be a useful but rather complex and involved method for evaluating the performance of the H6000 system, the major problem being the amount of work needed to count manually the number of cells required for the manual reference method.     

ENUMERATING 3MTM PETRIFILMTM AEROBIC COUNT PLATES USING THE PETRISCAN® AUTOMATED COLONY COUNTER

In the food and dairy industries, aerobic plate counts are determined by a time-consuming and laborious hand-counting method. The PetriScan ® automated colony counter was developed to improve efficiency in the microbiology laboratory. In this study, colony counts of food, dairy, and milk products plated on 3M^TM Petrifilm^TM Aerobic Count Plates were compared using both automated and manual count plate methods. For sample variation, 16 different food, dairy, and milk products were used. Samples were prepared and serially diluted using Butterfield's diluent according to approved AOAC methods and APHA's Standard Methods. Plates were inoculated, incubated, and counted according to AOAC methods. For data collection, plates with counts between 5 and 300 colonies were included. A total of 55 low (5–30), 29 medium (31–100), and 23 high (101–300) count plates were used. Duplicate results were recorded for both methods; hand counts were tallied by two scientists. The duplicates of the mean log values for manual counts varied by 0.0005 and 0.0007, and the duplicates for the automated counts varied by 0.0011. The mean log value difference between the automated and manual counts for pooled data was 0.035. The correlation coefficient for the regression line comparing the automated and manual count methods for pooled data was 0.98. The regression equation was y = 0.9257x + 0.0781. These results demonstrate that the PetriScan® automated colony counter is a comparable and practical alternative to the standard method of manually counting plates.     

Effect of shiftwork on systemic markers of inflammation

Shiftwork is associated with an increased risk of cardiovascular diseases, but the possible role of inflammation in this relationship is not well known. We tested the hypothesis that shiftwork would be associated with higher levels of C-reactive protein (CRP) and increased leukocyte count. We analyzed the cross-sectional associations between work arrangements and low-grade inflammation in 1877 airline-company employees separately for men (n?=?1037) and women (n?=?840). The participants were classified into five categories according to their work schedule: day workers who have not worked in shifts (referent group), former shiftworkers, 2-shift workers, 3-shift workers, and in-flight workers. In models adjusted for age and recent infectious diseases, CRP levels were higher among male 3-shift workers (p = .002) and marginally higher in male 2-shift workers (p = .076). In addition, leukocyte count was higher in 2-shift (p = .005) and 3-shift (p = .021) working men. In women, CRP level was higher in 2-shift workers (p = .028), whereas leukocyte count was lower in flight workers (p = .005). Any separate adjustment additionally for smoking, education, alcohol consumption, physical activity, and obesity did not substantially affect the results of 2- and 3-shift work. In the fully adjusted model, only the association between 3-shift work and CRP in men (p = .021) and 2-shift work and leukocyte count in men (p = .020) and leukocyte count in 3-shift-working women (p = .044) were significant. Our results suggest that 2- and 3-shift work is associated with increased systemic inflammation and the relationship is relatively independent of the considered risk factors of cardiovascular disease.     

Leukocyte Profiles in Wild House Finches with and without Mycoplasmal Conjunctivitis,a Recently Emerged Bacterial Disease

Leukocyte profiles (relative numbers of white blood cell types) have been used by a growing number of ecological studies to assess immune function and stress in wild birds. House Finches (Carpodacus mexicanus) in eastern North America are susceptible to an eye disease caused by the bacterium Mycoplasma gallisepticum, providing the opportunity to examine whether leukocyte profiles are associated with infection status and other host characteristics. In this study, we compared blood smears from 297 wild House Finches with and without conjunctivitis to examine whether leukocyte profiles were associated with the presence and severity of mycoplasmal conjunctivitis. We also evaluated the influence of age, sex, and molt on leukocyte profiles in both diseased and nondiseased birds. Of 243 House Finches of known age and sex sampled, 30% showed clinical signs of mycoplasmal conjunctivitis. House Finches with conjunctivitis had significantly higher heterophil to lymphocyte (H/L) ratios and harbored greater numbers and proportions of heterophils and monocytes than nondiseased birds. Leukocyte profiles of noninfected birds did not differ significantly with respect to sex, but young birds had significantly greater numbers of lymphocytes and total white blood cells than adults. Molting birds had significantly more eosinophils than nonmolting birds. Finally, House Finches with the most severe outward signs of conjunctivitis showed the most dramatic leukocyte changes relative to noninfected individuals, and increasing H/L ratios and monocytes in diseased birds were paralleled in a subset of birds that were recaptured during the study period. These results are consistent with patterns observed in domestic poultry and suggest that understanding patterns of leukocyte differentials in this host-pathogen system could improve our understanding of innate immunity and infectious disease risk in other wild passerines.     

Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: correlation with colony assay

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.     

Elevated cerebrospinal fluid leukocyte count and protein concentration at diagnosis: independent risk factors in children with acute lymphoblastic leukemia

The aim of this study was to investigate whether determination of the initial cerebrospinal fluid (CSF) protein concentration and leukocyte count in children with acute lymphoblastic leukemia (ALL) could yield useful information about the patient's central nervous system status and prognosis. The population-based unselected series comprised 160 children. The mean follow-up time was 72 months (range 25-143 months). Both the CSF protein concentration and the leukocyte count, if elevated, were significantly, although not independently, associated with diminished probability of event-free survival. The patients were divided into three groups for the final analyses: those without any abnormalities in the CSF (n = 133), those with elevated protein concentration and/or elevated leukocyte count, but with no malignant lymphoblasts in the CSF (n = 21), and those with malignant lymphoblasts in the CSF (n = 6). The probabilities of 5-year event-free survival for the first and second group were 65% and 15%; the probability of 2-year event-free survival for the third group was 17%. These differences were statistically significant (p less than 0.001). In multivariate analysis the relative risks of death or relapse for these groups were 1, 2.8 (95% confidence limits 1.5-4.9), and 7.6 (2.4-24.3), respectively (p less than 0.001). The inclusion of an elevated CSF protein concentration or leukocyte count in the risk group criteria of further trials should be considered.     

Evaluation of an Automated Colony Counter

An automated colony counter was found to readily detect surface and subsurface bacterial colonies of 0.3-mm size or greater with a high degree of precision. On a logarithmic scale, counting efficiency consistently ranged from 89 to 95% of corresponding manual count determinations for plates containing up to 1,000 colonies. In routine application, however, automated plate counts up to approximately 400 colonies were selected as a more practical range for operation. The automated counter was easily interfaced with an automated data acquisition system.     

温室白粉虱自动计数技术研究初报

应用计算机视觉技术对温室白粉虱自动计数技术进行了研究。采用胶卷照相机和家用摄像机对田间温室白粉虱等生的叶片进行拍摄,以获得其数字图象,对白粉虱图象的分割采用Ｊohannsen基于熵的分割算法,对分割后的二值图象利用区域标记算法得到白粉虱个体的数量。对叶片挨在一起的白粉虱个体采用数学形态学算法进行了分离。用１９个虫叶片样本的统计结果表明,直接利用分割图象进行白粉虱个体计数的累积准确率达９１．９９％,而分离处理的算法则需要改进,因此,这一技术具有进一步在生态研究和ＩＰＭ实践中推广的可能性,这将使田间微小昆虫的种群数量监测和测查的工作量大幅度降低,而铉得到显著提高。     

Automated topographical cell proliferation analysis

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.     

Acid glycosidases of normal cattle lymphocytes and in chronic lympholeukemia

The activity of five acid glycosidases was determined in lymphocytes from normal animals and animals with chronic lymphocytic leukemia. It was shown that alpha-D-mannosidase activity in leukemic lymphocytes was 5 times lower (p less than less than 0.001) and alpha-D-glucosidase activity was 2 times lower (p less than 0.01) than in normal controls. The progress of the disease and the increase in leukocyte count were accompanied by the decrease in alpha-D-mannosidase activity. No differences have been found in alpha-D-mannosidase properties (thermostability, Km values, ZnSl2 activation) in normal and leukemic lymphocytes.     

Sedative and cardiovascular effects of midazolam in swine.

The ability to reliably produce sedation in swine is hampered by the paucity of agents available. This project examined the use of a new water soluble benzodiazepine, midazolam, as a sedative in swine. Echocardiographic studies were performed on thirty 23 to 30 kg Yorkshire swine before and 20 minutes after each animal received a single intramuscular dose of 100 micrograms/kg midazolam. Heart rate and respiratory rate decreased significantly compared to nonsedated values (93 +/- 7 versus 117 +/- 2 bpm and 10 +/- 1 versus 20 +/- 1 breaths/min, respectively [p less than 0.05]). However, there was no effect on left ventricular fractional shortening (29.9 greater than 0.05 versus 29.5 +/- 0.05% [p greater than 0.05]). An additional five pigs were instrumented for a dose response study in order to collect hemodynamic data and blood gas values at baseline, and 15 min after the intravenous administration of incremental doses of midazolam (100 to 1,000 micrograms/kg). Despite a significant decrease in heart rate and respiratory rate, cardiac output, blood gases, and pH remained within normal ranges at all dosage levels. Both routes of administration produced sedation for 20 min in all animals. Midazolam is an effective swine sedative that is associated with stable cardiac function.