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PubMed2002

Reticulocyte analysis by flow cytometry and other techniques.

Enumeration of peripheral blood reticulocytes is an essential part of the diagnosis and management of anemic patients, since the number of reticulocytes in the peripheral blood reflects the erythrocytic activity of the bone marrow. Reticulocyte enumeration using flow cytometric methodology is rapidly replacing the inaccurate, imprecise manual counting technique used in the past. This article explores the pathophysiology of the reticulocyte, the various means of counting reticulocytes, and the diverse clinical applications of reticulocyte data.

Antibodies, Monoclonal↗
PubMed2005

Individual variation in CD4 cell count trajectory among human immunodeficiency virus-infected men and women on long-term highly active antiretroviral therapy: an application using a Bayesian random change-point model.

The authors evaluated population- and individual-level CD4-positive T-lymphocyte (CD4 cell) count trajectories over a 7-year period (July 1995-March 2004) following initiation of highly active antiretroviral therapy (HAART) in the Multicenter AIDS Cohort Study and the Women's Interagency HIV Study. The study population included 404 human immunodeficiency virus (HIV)-infected men and 609 HIV-infected women who 1) had a CD4 cell count measurement available from their last pre-HAART study visit, 2) provided at least four post-HAART CD4 cell count measurements, and 3) reported HAART usage for at least 80% of the post-HAART visits. The CD4 cell count trajectory was analyzed by means of a Bayesian random change-point model. The results indicated that CD4 cell count trajectories for long-term frequent HAART users can be well modeled with change points at both the population and individual levels. At the population level, regardless of CD4 cell count before HAART initiation, the gains in CD4 cell count ended approximately 2 years after HAART initiation in both men and women. At the individual level, 35% of men in the Multicenter AIDS Cohort Study versus 25% of women in the Women's Interagency HIV Study had a statistically significant change in CD4 cell count trajectory within 7 years after HAART initiation.

Adult↗
PubMed2006

Computational Methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells.

Observation and counting of gamma-H2AX foci in untreated cells as well as in cells exposed to cytotoxic agents is a widely used method for documenting the presence of double-strand breaks (DSBs) in the DNA and for analysis of their repair. Similar methods are employed to analyze formation of foci by a variety of proteins implicated in the cellular responses to DNA damage. Despite the wide application of the approach, the manual counting that is frequently used is prone to inaccuracies and investigator-related biases and artifacts. To alleviate this limitation, we developed and describe here personal computer-based algorithms, operating as utilities on available software, that allow an objective and quantitative analysis of foci from confocal images. The algorithms allow focus counting as well as size definition and correct for focus coincidence due to the overlap normally occurring with an increasing number of foci per nucleus. Furthermore, the software allows measurement of the integrated optical density (IOD) of each individual focus, which enables analysis of properties of foci as a function of time. Finally, the information generated by the above analysis algorithms can be employed to evaluate colocalization between foci formed by different proteins. A validation of the software is presented for radiation-induced gamma-H2AX foci in three widely used human cell lines and colocalization tested with RAD51 and gamma-H2AX foci. The computational methods presented extend to images generated by digital cameras.

Artificial Intelligence↗
PubMed2000

Bacterial chromosomal painting for in situ monitoring of cultured marine bacteria.

We previously described a new method, bacterial chromosomal painting (BCP), for the in situ identification of bacterial cells. Here, we describe the application of this technique to study the ecology and physiology of cultured marine pelagic bacteria from the western Sargasso Sea (WSS). A total of 86 bacteria were isolated from seawater collected from near the surface, at a depth of 250 m and from nutrient-amended seawater incubations. The 10 bacterial isolates that were best represented in environmental genomic DNA from the WSS were selected using reverse genome probing. BCP hybridization cell counts were used to determine the depth-specific distribution of one of the alpha proteobacterial isolates, B5-6, in the WSS during two thermal stratification regimes: stratified and partially mixed. The maximum cell count measured for B5-6 at the summer deep chlorophyll maximum was approximately 4% of the total cell count. This study is the first application of BCP to natural environments.

Alphaproteobacteria↗
PubMed2005

Percutaneous application of peptidoglycan from Staphylococcus aureus induces an increase in mast cell numbers in the dermis of mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease with immunopathologic features that vary depending on the duration of the lesion. The dermis of lesional skin of AD patients shows an increased number of inflammatory cells such as mast cells, eosinophils and mononuclear cells and superficial Staphylococcus aureus colonization. OBJECTIVE: The purpose of this study was to determine the effects of peptidoglycan (PEG) from S. aureus on mast cell induction in murine skin. METHODS: PEG was applied to barrier-disrupted abdominal skin of mice every 5 days and the number of mast cells in the abdominal skin was counted 20 days after the first application. The cytokine response was investigated by RT-PCR and immunohistologic analysis. RESULTS: The number of mast cells in the skin of mice treated with PEG was increased significantly compared with that of mice given phosphate-buffered saline. In addition, application of PEG to the abdominal skin increased the expression of mRNA for transforming growth factor-beta(1) (TGF-beta(1)), which supports mast cell migration, but not that for IL-3 or stem cell factor, which support both mast cell proliferation and mast cell migration. Immunohistologic analysis demonstrated that levels of TGF-beta(1) transcripts corresponded with those of protein synthesis in the epidermis. TGF-beta(1) was found to be highly expressed in keratinocytes of the basal epidermis of PEG-treated skin. Furthermore, intraperitoneal injection of anti-TGF-beta(1) antibodies neutralized the induction of mast cells into the skin. CONCLUSION: These results suggest that PEG may have the ability to induce an increase in mast cell numbers in the skin through TGF-beta(1) production by epidermal keratinocytes. Skin inflammation might therefore be linked to colonization with S. aureus in AD patients.

Abdomen↗
PubMed1996

Effects on locomotor activity after local application of (+)-UH232 in discrete areas of the rat brain.

The preferential dopamine autoreceptor, and slightly D3 preferring, antagonist (+)-UH232 (cis-(+)-(1S,2R)-5-methoxy-1-methyl-2-(n-dipropylamino) tetralin) increases locomotor activity and synaptic dopamine release in the nucleus accumbens and striatum after systemic administration to the rat. As shown in this study, (+)-UH232, was unable to produce an increase in locomotor activity measured for 60 minutes after local administration into the terminal or somato-dendritic regions of the mesolimbic dopamine pathways or into the lateral ventricle. Instead, a dose dependent decrease of spontaneous locomotor activity after local application (0.05-50.0 nmol/side) of (+)-UH232 into the nucleus accumbens, was seen. A similar reduction in locomotor activity was produced by the classical dopamine antagonist raclopride. Analysis of the dose*time interactions on locomotor activity did, however, indicate that there is a significant dose*time interaction after local application of (+)-UH232 into the lateral ventricle and VTA. Raclopride, on the other hand, produced only a weak time dependent effect in the VTA. The potential problem of Leao's spreading depression in micro-injection experiments were considered, however, spreading depression does not seem to influence the effects of (+)-UH232 locally applied into the nucleus accumbens. In conclusion, both (+)-UH232 and raclopride produced a dose dependent decrease in spontaneous locomotor activity when examined as the total activity count over 60 minutes after local application into the N Acc.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1996

Interference of blood leucocytes in the measurements of immature red cells (reticulocytes) by two different (semi-) automated flow-cytometry technologies.

Flow cytometrical methods have been introduced recently as an alternative to the enumeration of reticulocytes by microscopy. Two of these methods have gained widespread use in haematological practice; the multiparametric flow cytometer using thiazole orange staining (Retic-Count, FACScan) and the single-application reticulocyte counter using auramine-O staining (R-series, Sysmex). Several studies have emphasized the excellent correlations between microscopy and these techniques. The purpose of our study has been to examine the specificity of these automated devices with regard to cells classified as 'reticulocytes' and the effect that this may have on measures of reticulocyte maturity. Our results indicate that the specificity of reticulocyte measurements by both the Sysmex R-1000/-3000 and the Retic-Count system is relatively low. This is due to the presence of leucocytes amongst cells classified as reticulocytes. These leucocytes display intense staining with either dye, leading to an erroneous estimation of RMI (thiazole orange) and high fluorescence count (R-1000/-3000). This error is directly correlated with the leucocyte count. The basis for reticulocyte identification should be improved before automated estimation of reticulocyte maturation can be used in clinical practice.

Automation↗
PubMed1996

The gender-specific chest wall thickness prediction equations for routine measurements of 239Pu and 241Am within the lungs using HPGe detectors.

The current chest wall thickness prediction equation is not applicable to use in routine lung counting measurements for detection of low energy photons (17--60 keV) within the lungs of male and female subjects. The current chest wall thickness prediction equation was derived for the NaI-CsI "phoswich" detection system, which is not the routine detection system in use; the subject position was supine, which is not the routine position; the equation did not account for the intercostal tissue thicknesses of muscle and adipose which significantly attenuate low energy photons (17--60keV); it was derived from male subjects only and is used to predict the chest wall thickness of female subjects for whom it is not applicable. The current chest wall thickness prediction equation yields unacceptable percent errors in the HPGe detection efficiency calibration for 239Pu and 241Am (17- and 59.5-keV photons, respectively) relative to the gender-specific HPGe chest wall thickness prediction equations of this paper (+284% to --73% for 239Pu; (+)42% to --39% for 241Am). As a result, use of the current chest wall thickness prediction equation yields unacceptable percent errors (proportional in magnitude to the percent errors in the detection efficiency calibration) in the calculation of the minimum detectable activity (Bq) or in an initial assessment of a radioactive contamination exposure detected by a routine lung count measurement.

Adult↗
PubMed2005

On-line counting of cysteine residues in peptides during electrospray ionization by electrogenerated tags and their application to protein identification.

The electrochemically induced mass spectrometric tagging of cysteines by substituted hydroquinones was studied for peptides in a classical electrospray solvent (i.e., MeOH/H2O/AcOH 50/49/1). The tagging efficiency was tested with different hydroquinone compounds on an undecapeptide containing one cysteine residue. 2-carboxymethylhydroquinone was the most reactive probe and revealed to be suitable for cysteine quantification in peptides containing up to three cysteine residues, even in the case of two consecutive cysteines in the sequence. We demonstrate the compatibility of the on-line electrochemical tagging method for the cysteine content analysis of peptides coming from gel-free protein digestion procedures. The identification of bovine serum albumin and human alpha-lactalbumin digest samples in a peptide mapping strategy was greatly improved by the application of the electrotagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provided powerful information in order to enhance the identification score as well as the discrimination against other protein candidates. The tagging method was then applied to the determination of four proteins in a model mixture.

Animals↗
PubMed2005

Comprehensive language mapping in children, using functional magnetic resonance imaging: what's missing counts.

Noninvasive language mapping is a frequently used application of functional magnetic resonance imaging. The examination of children, however, poses both practical and technical challenges. To this effect, we have developed two new paradigms mainly requiring passive listening to stories from which several key words were removed. Results were compared with a standard active verb-generation task, particularly with regard to hemispheric dominance in frontal brain areas. Fifteen healthy children (seven boys, eight girls, 6-14 years) were scanned. Distinct frontal activation was seen in our new tasks, which was stronger when the tasks were analyzed specifically with regard to the missing words. This confirms the hypothesis that the removal of these words is the key in inducing frontal activation. Frontal lateralization was consistent and strong in all three paradigms. Additionally, receptive language areas are also mapped by our new tasks. We conclude that our two new, child-friendly functional magnetic resonance imaging paradigms are useful in determining hemispheric dominance for language in children too young or too impaired to execute an active task. They also allow robust mapping of receptive language areas.

Adolescent↗
PubMed1982

[Measurement of platelet volume: methodologic approach and application in diabetes].

A comparative study of platelet count and platelet volume distribution was performed on whole blood (Coulter counter S+) or platelet rich plasma (Thrombo Coulter and Coulter ZBI + Channelyser C 1 000). With the exception of severe thrombocytopenia the results of platelet counts obtained with the coulter S + were very reproducible (CV less than 4%) and a good correlation was observed between results obtained on whole blood and on platelet rich plasma (PRP). The mean platelet volume (MPV) was significantly higher in whole blood than in PRP, but the difference was very slight, suggesting that platelet volume distribution in PRP and in whole blood are similar. In whole blood collected in EDTA MPV increased significantly during the 2 hours after blood collection and then reached a plateau. This finding underlines the need for a standard technique. Preliminary results obtained with the Coulter S + in 86 diabetic patients and in 73 unselected control subjects demonstrated a significant increase of MPV and of the index of platelet distribution (IDP) given by the apparatus, without modification of platelet count in diabetics. These results could suggest a compensated increased platelet consumption in the circulating blood.

Blood Platelets↗
PubMed1998

A computer-aided 3-D geometry of acute and chronic zonal necrosis: three-D tangent counting applied in an attempt to re-examine the structure of the human liver.

In an attempt to examine the feasibility of the acinar concept in the liver of humans, the spatial distribution of zonal necrosis and its relation with the blood vessels were studied in terms of 3-D tissue microstructure. The material was five autopsy livers, two from patients of acute cresol intoxication and three with long-standing heart failure. Examined were: 1) whether the surviving areas (zones 1, 2) in acute zonal necrosis have surfaces convex toward the necrotic zone 3 as illustrated in the acinar schema, and 2) whether the necrosis extends as slender "s eeves" so as to reach the portal tracts and create portal-central (P-C) bridging, leaving zones 1 and 2 as a spheroid parenchymal clump. These were examined on serial histological sections of the livers, which were subjected to 3-D reconstruction with the aid of a computer system. The geometric properties of the interfaces between the surviving and necrotic areas were analyzed by 3-D tangent counting technique of DeHoff modified for 3-D application. It was shown by reconstruction that necrosis was uniformly distributed around the hepatic venules. In acute cases, tangent counting showed that the surfaces of the surviving areas were mainly concave toward necrosis. In contrast, in chronic cases, the surfaces became mainly convex as a result of parenchymal regeneration. At no place, in acute or chronic cases, was necrosis shown to reach the portal tracts, and because of this, neither acini nor their agglomerates revealed themselves as a separate unit. All these findings contradict what one expects would be, so long as based on the assumption of the acinar model.

Acute Disease↗
PubMed1989

A dose-dependent effect of recombinant erythropoietin on the reticulocyte population of rats.

The effect of human recombinant erythropoietin (rEPO) on the reticulocyte count and the reticulocyte maturation distribution in rats was measured with an automatic reticulocyte analysis system (AURAS). The strongest reaction was found on the third day after a single application of rEPO with respect to reticulocyte count, maturation distribution, and the count of immature reticulocytes. At this time these parameters show a distinct dose dependency. The results indicate a possible basis for a simple rEPO bioassay using normocytemic rats.

Animals↗
PubMed2003

Determination of potential dermal and inhalation operator exposure to malathion in greenhouses with the whole body dosimetry method.

One of the steps during the authorization process of plant protection products (PPP) in the European Union is to evaluate the safety of the operator. For this purpose, information on the probable levels of operator exposure during the proposed uses of the PPP is required. These levels can be estimated by using existing mathematical models or from field study data. However, the existing models have several shortcomings, including the lack of data for operator exposure levels during spray applications by hand lance, especially in greenhouses. The present study monitored the potential dermal and inhalation operator exposure from hand-held lance applications of malathion on greenhouse tomatoes at low and high spraying pressures. The methodology for monitoring potential exposure was based on the whole body dosimetry method. Inhalation exposure was monitored using personal air pumps and XAD-2 sampling tubes. For the monitoring of hand exposure, cotton gloves were used in two trials and rubber gloves in another three. The total volumes of spray solution contaminating the body of the operator were 25.37 and 35.83 ml/h, corresponding to 0.05 and 0.07% of the applied spray solution, respectively, in the case of low pressure knapsack applications and from 160.76 to 283.45 ml/h, corresponding to 0.09-0.19% of the spray solution applied, in the case of hand lance applications with tractor-generated high pressure. Counts on gloves depended on the absorbance/repellency of the glove material. The potential inhalation exposures were estimated at 0.07 and 0.09 ml/h in the case of low pressure knapsack applications, based on a ventilation rate of 25 l/min. Both potential dermal operator exposure (excluding hands) and potential inhalation exposure were increased by a factor of approximately 7 when the application pressure was increased from 3 to 18 bar in greenhouse trials with a tractor-assisted hand lance, the rest of the application conditions being very similar.

Agriculture↗
PubMed2005

Rapid counting of liquid-borne microorganisms by light scattering spectrometry.

Fast and sensitive techniques are needed to determine microorganism presence in liquid samples. In this research, the feasibility of using light scattering spectrometry for enumerating the biological particles in liquid samples was investigated. A particle size spectrometer was used to count six commonly found microbial species suspended in liquid with and without microbiological stains applied: Pseudomonas fluorescens, Micrococcus spp. vegetative cells and Bacillus subtilis var. niger endospores were stained with Acridine Orange and Crystal Violet, while Cladosporium cladosporioides, Penicillium melinii and Aspergillus versicolor fungi were stained with Acridine Orange and Lactophenol Cotton Blue. The counts obtained with the spectrometer were compared with those obtained with a phase-contrast microscope. It was found that the spectrometer counted about 32 % of non-stained B. subtilis endospores and this percentage increased to almost 90 % for stained endospores. Among the investigated species of fungi, the counting efficiency of P. melinii was the only one significantly affected by the application of the stain Lactophenol Cotton Blue: the fraction of counted fungal spores increased from 64 % (non-stained spores) to about 100 % (stained spores). The observed difference in counting efficiency may serve as a basis for differentiating biological from non-biological particles in liquid samples.

Air Microbiology↗
PubMed1988

Importance of diagnostic aspects in ostertagism.

Diagnostic methods which reveal only the presence or absence of Ostertagia in grazing animals are of little importance since all will acquire some degree of infection when grazed in the temperate regions of the world. Ostertagia infections may be (1) light--no disease or performance losses occur; (2) moderate--disease may or may not be overtly visible but performance losses occur or (3) heavy--disease is overt with or without death loss. The differentiation of these levels of infection is a quantitative matter insofar as the number of parasites correlates with the degree of pathology and is much less certain than is the presence or absence of infection. Historically, the quantitative parasite egg count in feces (eggs per gram (e.p.g.)), combined with the usual subjective and objective information obtained in clinical examinations has been the procedure used to obtain a quantitative estimate of the level of infection. More recently, the use of plasma pepsinogen levels as an indication of gastric pathology has been adopted by some laboratories. Neither of these methods have met with the desired level of accuracy, but they do have considerable value when their limitations are recognized. In the present era of emphasis on epidemiologically based control programs we are faced with yet another aspect of diagnosis, namely that of a quantitative estimate of numbers of infective stages (L3) on herbage. Methods to assess the number of L3 on forage are not satisfactory for routine clinical use. This paper evaluates the primary objectives of diagnosis as applied to the herd and flock. It briefly evaluates current methodology insofar as the diagnosis of parasitic gastroenteritis is concerned. The use of e.p.g. counts in clinical practice is discussed. Examples applicable to herds of weaner and stocker cattle are presented.

Animals↗
PubMed1996

Cerebrospinal fluid values in the term neonate.

BACKGROUND: Cerebrospinal fluid (CSF) values in the noninfected neonate are not well-delineated. Studies analyzing these values are inconsistent in the criteria used to define the noninfected population. The purpose of our study was to examine CSF values in neonates in the first 30 days of life in whom infection was more thoroughly excluded than in previous reports. Stringent inclusion criteria defined the noninfected population, and the recently available polymerase chain reaction (PCR) for enteroviruses was used in addition to cultures to help exclude viral disease. Results were also stratified by age in weeks to evaluate for any variability that occurs in CSF values during the first month of life. METHODS: Neonates were selected from subjects enrolled in two studies on aseptic meningitis. Noninfected infants were identified by the following criteria: (1) atraumatic lumbar puncture (< or = 1000 red blood cells/mm3); (2) no antibiotic therapy before lumbar puncture; (3) sterile blood, CSF and urine bacterial cultures; (4) negative CSF viral culture; and (5) negative CSF PCR for enteroviruses. RESULTS: The mean +/- SD total CSF white blood cell count for 108 noninfected neonates was 7.3 +/- 14/mm3 (95% confidence interval 6.6 to 8.0/mm3) with a median of 4/mm3 and a range of 0 to 130/mm3. There were no significant differences in the mean CSF white blood cell counts among age categories. CONCLUSIONS: The application of stringent inclusion criteria and the use of the PCR yielded a population of infants that better represents the noninfected neonate than earlier reports. These values can be used for reference in evaluating the febrile or ill neonate.

Age Factors↗
PubMed2006

Effects of topical application of methotrexate on nasal mucosa in rats: a preclinical assessment study.

OBJECTIVE: The aim of this study is to investigate dose-dependent effect of the topical application of methotrexate (MTX) in rats on the normal nasal mucosa, liver tissue, liver enzymes, and hemoglobin levels. STUDY DESIGN: Preclinical animal study. SETTING: Twenty male adult wistar albino rats were randomly divided into 4 groups (n=5). A single puff of MTX (2.5 microg) was applied to both nasal cavities 2 times a day. The animals were given MTX 1 day a week in group 1, 3 days a week in group 2, and 5 days a week in group 3. Control group animals were given 1 puff of physiologic saline to both nasal cavities 5 days a week and 2 times a day. After 28 days, liver biopsies, blood samples, and 5 nasal mucosal biopsies were taken. Histological examination was made with respect to certain parameters semiquantitatively (grade 0-3). The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and hemoglobin counts were studied from blood samples. RESULTS: There are no statistically significant differences with respect to histopathological parameters between the control group and the groups 1-3 (P>0.05). Histopathological examination of liver tissue did not reveal any evident difference between the control and study groups. Mean AST and ALT as liver function tests and hemoglobin counts were within normal limits. Topical application of MTX at these doses has no toxic effect on the nasal mucosa, the liver tissue, AST and ALT levels, and hemoglobin level. CONCLUSIONS: These results have been encouraging to investigate use of the topical application of MTX in nasal manifestation of autoimmune disease or addition of the topical application of MTX to the steroid treatment in cases with massive nasal polyposis resistant to steroids and prone to recurrence.

Administration, Intranasal↗