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A reliable screening protocol for thalassemia and hemoglobinopathies in pregnancy: an alternative approach to electronic blood cell counting.

Primary screening for thalassemia and hemoglobinopathies usually involves an accurate blood count using an expensive electronic blood cell counter A cheaper alternative method was tested by using a modified osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) test. Altogether 423 pregnant Thai women participated in this project. Hemoglobin patterns and globin genotypes were determined using an automated high-performance liquid chromatography analyzer and polymerase chain reaction analysis of alpha- and beta-globin genes. Among the 423 subjects, 264 (62.4%) carried thalassemia genes. The combined OF and DCIP tests detected all pregnant carriers of the 3 clinically important thalassemias, ie, alpha0-thalassemia, beta-thalassemia, and hemoglobin E with a sensitivity of 100.0%, specificity of 87.1%, positive predictive value of 84.5%, and negative predictive value of 100.0%, which show more effectiveness than these values for the standard method based on RBC counts. A combination of modified OF and DCIP tests should prove useful and applicable to prenatal screening programs for thalassemia and hemoglobinopathies in communities with limited facilities and economic resources.

Adult↗

Influence of two different approaches to reporting implant survival outcomes for five different prosthodontic applications.

BACKGROUND: Over the years, the definition of implant failure has varied, with some investigators accounting for all implants placed, while others discard failures that occurred before clinical loading. The influence of stresses transmitted to various bone densities, by different prosthetic appliances as well as the method used to determine failures, needs to be clearly understood. This paper reports on the influence of two different methods of determining 36-month survival of implants used to support different dental prostheses. METHODS: More than 2,900 implants with six different designs were placed in 829 patients at 32 study centers and followed for 3 years. The first method of determining survival accounted for all failures from placement through 36 months, while the second method counted only failures from post-loading of the prosthesis to 36 months. Survival curves were used to determine differences in survival outcomes for the two methods. RESULTS: For the maxillary single-tooth prosthetic application, implant survival from placement to 36 months was 94.7% when all failures were counted and increased to 98.3% with the post-loading method. For upper completely edentulous applications, implant survival was 85.3% with all failures counted and 95.6% with the post-loading method. This 10.3% difference is clinically important. The survival for implants in lower completely edentulous applications increased by 4.4% simply by using the post-loading approach. Implants used for upper posterior, partially edentulous applications involved only hydroxyapatite (HA)-coated implants, and the survival rates were similar (96.4% for all implants and 98.2% for the post-loading method). The difference in reported survival rates for implants in the lower posterior, partially edentulous application was 5.8%. Since failures tended to occur in the earlier phases of treatment, the post-loading approach always resulted in more favorable survival data. With the post-loading approach, valuable information related to implant performance before loading is lost. In all comparisons, HA-coated implant survival was always better than non-HA implants. Clinical investigators should clearly state the method used for determining failures. For all implants included in the study, survival curves illustrated different failure patterns for each method of determining overall survival. CONCLUSION: Reporting of implant survival rates based on the post-loading method provides more favorable survival rates; however, accounting for all implants provides a more accurate method of determining survival.

Adult↗

"Virtual flow cytometry" of immunostained lymphocytes on microscopic tissue slides: iHCFlow tissue cytometry.

BACKGROUND: A method and approach is developed for fully automated measurements of immunostained lymphocytes in tissue sections by means of digital color microscopy and patent pending advanced cell analysis. The validation data for population statistic measurements of immunostained lymphocytes in tissue sections using tissue cytometry (TC) is presented. The report is the first to describe the conversion of immunohistochemistry (IHC) data to a flow cytometry-like two parameter dot-plot display, hence the technique is also a virtual flow cytometry. We believe this approach is a paradigm shift, as well as novel, and called the system iHCFlow TC. Seven issues related to technical obstacles to virtual flow cytometry (FC) are identified. DESIGN: Segmentation of a 512 x 474 RGB image and tabular display of statistical results table took 12-15 s using proprietary developed algorithms. We used a panel of seven antibodies for validation on 14 cases of mantle cell lymphoma giving percentage positive, total lymphocytes, and staining density. A total of 2,027 image frames with 810,800 cell objects (COBs) were evaluated. Antibodies to CD3, CD4, CD8, Bcl-1, Ki-67, CD20, CD5 were subjected to virtual FC on tissue. The results of TC were compared with manual counts of expert observers and with the results of flow cytometric immunophenotyping of the same specimen. RESULTS: The correlation coefficient and 95% confidence interval by linear regression analysis yielded a high concordance between manual human results (M), FC results, and TC results per antibody, (r = 0.9365 M vs. TC, r= 0.9537 FC vs. TC). The technical issues were resolved and the solutions and results were evaluated and presented. CONCLUSION: These results suggest the new technology of TC by iHCFlow could be a clinically valid surrogate for both M and FC analysis when only tissue IHC is available for diagnosis and prognosis. The application for cancer diagnosis, monitoring, and prognosis is for objective, rapid, automated counting of immunostained cells in tissues with percentage results. We report a new paradigm in TC that converts IHC staining of lymphocytes to automated results and a flow cytometry-like report. The dot plot histogram display is familiar, intuitive, informative, and provides the pathologists with an automated tool to rapidly characterize the staining and size distribution of the immunoreactive as well as the negative cell population in the tissue. This systems tool is a major improvement over existing ones and satisfies fully the criteria to perform Cytomics (Ecker and Tarnok, Cytometry A 2005;65:1; Ecker and Steiner, Cytometry A 2004;59:182-190; Ecker et al., Cytometry A 2004;59:172-181).

B-Lymphocytes↗

Ocular penetration of topical ciprofloxacin and norfloxacin drops and their effect upon eyelid flora.

A double blind, prospective study was undertaken to compare aqueous humour penetration of topical 0.3% norfloxacin and 0.3% ciprofloxacin and their effect upon normal eyelid flora in 39 patients undergoing cataract surgery. Lid swabs were taken before and after six 1 hourly applications of single drops of ciprofloxacin or norfloxacin given before surgery. Aqueous humour was aspirated at surgery and antibiotic concentration assayed using high performance liquid chromatography. The mean aqueous humour concentrations were: ciprofloxacin 220 ng ml-1, norfloxacin 140 ng ml-1. Although this difference was not statistically significant (p = 0.112) the trend demonstrated may be relevant clinically, especially considering the greater activity of ciprofloxacin. Both coagulase negative staphylococcal (p = 0.004) and total bacterial (p = 0.019) lid counts dropped sixfold after ciprofloxacin treatment but the smaller reductions noted after norfloxacin application did not achieve statistical significance (p > 0.1). The reduction of external eye flora experienced with ciprofloxacin suggests that this may be a useful presurgical prophylactic agent.

Administration, Topical↗

CD4 counts of normal and HIV-infected south Indian adults: do we need a new staging system?

BACKGROUND: The normal range of CD4 lymphocyte count varies among different ethnic groups. In populations with CD4 counts inherently lower than in the West, the Centers for Disease Control and Prevention (CDC) classification system of HIV-infected individuals may not be appropriate. No such criterion is currently available for ethnic south Indian HIV-infected individuals. We undertook this study to assess the applicability of the western case definition to south Indian HIV patients. METHODS: The CD4 counts of 104 normal and 79 HIV-infected south Indian adults were estimated by flowcytometry. The mean CD4 counts were estimated and compared between CDC groups A, B and C and controls. Receiver operator characteristics curves were generated to determine the cut-off that correlated best with clinical staging for this population. RESULTS: The mean CD4 and CD8 cell counts/microl and the CD4/CD8 ratio of the control group were 799.0, 919.6 and 0.94, respectively. For CDC groups A, B and C, the mean CD4 counts/microl were 454.4, 254.4 and 168; mean CD8 counts/microl were 1156, 1070.9 and 1191.3; and mean CD4/CD8 ratios were 0.44, 0.25 and 0.15, respectively. CONCLUSION: The mean CD4 count among normal south Indians is significantly lower than that in the western population and parallels that of the Chinese. When categorized based on the CDC classification system, the mean CD4 count of HIV-infected individuals was lower. Categories of CD4 counts > 300, 81-300 and < or =80 cells/microl and CD4% > 14, 7-14 and < or = 6 correlate better with disease progression among HIV-infected individuals. A longitudinal study is required before guidelines for the Indian population can be devised.

Adolescent↗

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Correspondence as Topic↗

Radiation biodosimetry: applications for spaceflight.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Becton [correction of Beckon] Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.

Animals↗

Automated malaria diagnosis using pigment detection.

Several new methods of malaria diagnosis have recently been developed, but these all rely on clinical suspicion and, consequently, an explicit clinical request. Although some methods lend themselves to automation (eg. PCR), no technique can yet be used for routine clinical automated screening. Detection of birefringent haemozoin has been used to diagnose malaria since the turn of the 20th century. A new generation of full blood count analysers, used widely in clinical laboratories, have the potential to detect haemozoin in white blood cells and probably erythrocytes. Thomas Hänscheid, Emilia Valadas and Martin Grobusch here describe this novel technique for malaria diagnosis and discuss its potential applications.

Blood Cell Count↗

Performance evaluation of the Pico-Count flow-through detector for use in cerebral blood flow PET studies.

UNLABELLED: This study evaluated the Pico-Count (Bioscan, Inc., Washington, DC) flow-through radioactivity detector, designed for use in PET studies of cerebral blood flow. METHODS: The Pico-Count detects the two 511-keV positron annihilation photons with two bismuth germanate detectors operating in coincidence. The detectors, photomultipliers and preamps are housed within a 12 cm x 9 cm x 22 cm box, which includes 16 mm of lead shielding, to allow placement of the detector within 15 cm of the sampling site. The counting electronics are housed in a remote box, which is connected to a laptop computer for process control. The dwell time per sample and the number of samples to collect are entered through the computer and can vary throughout the study. Approximately 22 cm of arterial tubing (which contains 0.11 ml of blood) is looped between the detectors. Typically, blood is withdrawn with a syringe pump at a rate of 2.75 ml/min, which corresponds to a flow rate in the tubing of 9.2 cm/sec. Dispersion within the arterial catheter is measured by observing the response to an input step function and is well-modeled as a monoexponential. RESULTS: The sensitivity is 270 Hz/(microCi/ml), which corresponds to detecting 6.9% of the positron decays occurring within the detector. The peak counting rate after a 12-mCi injection is approximately 2100 Hz, with the background being less than 0.2%. The dispersion time constant is 1.3 sec, and the delay between radioactivity present at the catheter tip and that measured by the detector is 4.1 sec. The cutoff in the power spectral density of typical human arterial blood time radioactivity curves is far less than the corresponding cutoff for the dispersion function. CONCLUSION: The Pico-Count is an excellent detector for continuously monitoring positron radioactivity in blood. Depending on the application, dispersion correction for the detection apparatus may not be needed.

Brain↗

Validity of existing CD4+ classification in north Indians, in predicting immune status.

BACKGROUND: The CD4 lymphocyte count had ethnic variability as observed in many studies. In populations with CD4 counts inherently lower than in the West, the Center for Disease Control and Prevention (CDC) classification system of HIV-infected individuals may not be appropriate. As there is no such criterion currently available for ethnic north Indians HIV-patients, we undertook this study to assess the applicability of the western case definition in north Indian HIV patients. METHODS: The CD4 counts of 40 normal and 376 HIV-infected north Indian adults attending to ID clinic, SS hospital, Varanasi were estimated by flowcytometry. The mean CD4 counts were estimated and compared between CDC groups A, B and C and controls. Receiver operator characteristic (ROC) curves were generated to determine the cut-off that correlated best with clinical staging for this population. RESULTS: For CDC groups A, B and C, the mean CD4 counts/mul (upper limits of the 95% CI) were 380.3, 249.2 and 120.9, while the mean CD4 levels in healthy volunteers was 818.4. CONCLUSION: The mean CD4 count among normal north Indians is significantly lower than that in the western population and parallels that of the Chinese. When categorized based on the Center for Disease Control and Prevention (CDC) classification system, the mean CD4 counts in HIV-infected individuals was lower. Categories of CD4 counts >280, 120-280 and < or =120 cells/microl correlate better with disease progression among HIV-infected individuals. A longitudinal study is required before guidelines for the India population can be devised.

Adult↗

[Synapse density determination in brain structures based on phosphotungstic acid contrast].

Using the E-PTA procedure of Aghajanian and Bloom we determined the numerical density of synapses in the stratum lacunosun-modeculare of the CA1-region in the hippocampus of male albino rats. On this occasion the sources of error both the natural and the methodical were investigated. As natural causes of remarkable scattering and failing correspondence in the findings the individual variability of the synaptic density and the inhomogeneous distribution of synapses must be taken into consideration. Concerning the methodical sources of error the differences in tissue shrinkage become apparent. But faults resulting from applying morphometric methods are the most serious. The counting of synaptic profiles is based on defined morphological criteria. The portion of the synaptic profiles recorded in this way in relation to all those present in the section depends beside other things on the section thickness. Accordingly the correction factors are in correlation with section thickness. The application of the Floderus correction presupposes the determination of the minimum length of the counted profiles. As a result of these investigated the possibilities for reducing the error have been demonstrated.

Animals↗

Fetal hematology.

The objective of this study was to establish normal fetal hematological parameters throughout gestation. Samples of pure fetal blood from 35 fetuses of 21-38 weeks' gestation were obtained by fetal blood sampling under continuous ultrasound guidance. The hematological parameters were determined with automated cell counter within 30 minutes after the procedures. Fetal red blood cell and granulocyte counts rose significantly with advancing gestation, whereas, the mean corpuscular volume fell. There were no significant changes in fetal hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, total white blood cell count, lymphocyte count, mid-cell count, platelet count, mean platelet volume, and platelet distribution width with increasing gestation. The growing application of fetal blood sampling to the prenatal diagnosis renders mandatory a knowledge of normal fetal blood values. These results may provide useful reference values for prenatal diagnoses of hematological disorders.

Blood Cell Count↗

Tick control on eland (Taurotragus oryx) and buffalo (Syncerus caffer) with flumethrin 1% pour-on through a Duncan applicator.

Eland (Taurotragus oryx) and buffalo (Syncerus caffer) in the Game Ranching Research Station at the Mushandike Sanctuary in Zimbabwe were treated with 1% flumethrin pour-on to control unacceptably high tick numbers. The pour-on was at first applied with a drenching gun and later by means of a Duncan Applicator. This device allows contact with a saturated treatment column while the animals consume a specially formulated attractant lick from the feed bin. Tick counts done over 3 summer seasons demonstrated the efficacy both of the pour-on acaricide and the method of application.

Animals↗

Simple markers for initiating antiretroviral therapy among HIV-infected Ethiopians.

BACKGROUND: We explored the relevance of simple markers (clinical or laboratory markers not requiring sophisticated laboratories) in the decision of initiation of therapy in resource-poor settings. METHODS: Among HIV-infected Ethiopian cohort participants, simple markers predicting short-term death were examined using time-dependent Cox proportional hazards models. Timing of hypothetical treatment was compared between guidelines using the simple markers (based on presence of at least one marker), guidelines recommended by the United States Department of Health and Human Services (based on CD4 cell count and viral load), and guidelines for resource-limited settings recommended by the World Health Organization (WHO). RESULTS: From February 1997 to August 2001, 35 deaths were recorded among 155 HIV-positive participants. Simple independent predictors of death were low body mass index, HIV-related conditions, anaemia, and lymphocyte count < 1500 x 106/l. In such time as was covered by our study, 135 (87%) of 155 cohort participants would have had the same management under both the simple markers and the DHHS guidelines, i.e., would have been treated (n = 114, 74%) or not treated (n = 21, 14%). Of the 114 participants hypothetically treated under either set of guidelines, 91 (80%) would have started treatment at the same time. Application of the WHO guidelines for resource-limited settings (without CD4 cell counts) would have resulted in 11 participants dying without ever meeting a treatment indication during regular follow-up visits. CONCLUSION: Simple markers for the initiation of highly active antiretroviral therapy were identified among HIV-infected Ethiopian patients. The validity of these markers for monitoring patients' improvement following therapy remains to be evaluated.

Adult↗

Morphometric applications in anatomic pathology.

The components of the cell and tissue changes in many diseases are variable and can therefore be quantified. Characterization of these quantitative changes provides data that is useful not only for making a definitive, cell- and tissue-based diagnosis of disease, but also for predicting the course of disease. The spectrum of changes found in malignant tumors, ie, cell grade, architecture, cellularity, extent of invasion, nature and extent of inflammatory reaction, exemplify this range of quantifiable features. The diagnosis and prognosis of nonneoplastic diseases, ie, myopathy and metabolic bone disease, can also be determined by quantitating tissue changes. Morphometry is the quantification of changes in the "objects" of tissues, ie, cells and organelles, and their organization, using quantitative evaluation tools. The principles of morphometry have been known for a century. With the increasing availability of affordable, powerful computer systems and increasingly flexible and user-friendly software has come easier ability to measure these changes. This article discusses the principles of morphometry with illustrations of types of analysis (ie, area fraction, object counting, shape and size analyses, and mutliparametric analyses) using examples of these applications with discussions of error sources and limitations of morphometry.

Cell Size↗

Automatic measurement of drinking in rats: effects of hypophysectomy.

A new apparatus for the continuous measurement of drinking in the rats was assembled. The principle of the device is as follows: a cartridge which makes water drops (0.05 ml) is inserted between a water tank and a drinking spout. When a rat drinks, water falls into the cartridge drop by drop and the number of drops is electrically counted. The total count of drops per day, as well as counts at definite intervals, can be atuomatically printed out. To test apparatus reliability and applicability, drinking behavior in hypophysectomized rats was investigated in the light and dark phases, alternating every 12 hr. Activity and feeding in these phases were also observed. In the sham-operated rats, the total daily water intake was 30-40 ml, which corresponded to 10-15% of the body weight, and 85-95% of the total daily drinking counts were recorded in the dark phase. In the hypophysectomized rats, a large amount of water was drunk immediately after the operation. However, the high rate of drinking rapidly returned to near the normal level within a few days. Drinking in the dark phase decreased to about 75% of the total daily, but synchronization with the light-dark cycle was still maintained. The daily patterns of activity and eating ran nearly parallel with the drinking behavior. These results indicate that our drinkometer could have extensive applications within many fields of research.

Animals↗