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PubMed1983

Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting.

Published values for the number of neurons in the superior cervical ganglion of the adult rat range from 13 000 to 45 000. These studies have employed different methods for determining what unit to count (cell body, nucleus, nucleolus), how many sections to count, and how to correct the raw counts for split particles and for profiles that are too small to resolve. The purpose of this study was to examine the extent to which these parameters may influence the calculated value for the total number of neurons, using computer simulations of neuron populations. These simulations permitted us to determine the effects on neuron number of varying the diameter of the neuronal nucleus, the size of the smallest resolvable profile, and the thickness of the section. The data from the simulations were used to test the validity of several methods that are in common use for correction of neuron counts. Our results indicate that most of the methods that are in routine use are unsatisfactory. We propose the use of either one of two methods that consistently result in highly accurate estimates of neuron numbers. These are: (1) a modification of the method proposed by Hendry (1976), using computer analysis; or (2) a modification of the method proposed by Abercrombie (1946), which does not require the use of a computer.

Animals↗
PubMed1990

A new development in haematological cell counting: the Sysmex NE-8000, automaton for cell count and physical five-part leukocyte differentiation.

We compared the results of the Sysmex NE-8000 with those of the Sysmex E-4000 and the Technicon H-6000. Both for the whole blood cell count and the leukocyte differentiation we obtained intra- and inter-assay coefficients of variation fully comparable with those for other cell counters. The carry-over between samples was negligibly small. The linearity range was broad, which implies a universal application in routine haematological analysis. The numerical results from the Sysmex NE-8000 and the Technicon H-6000 corresponded closely, with exception of the mean corpuscular volume and the haematocrit, whereas these parameters showed excellent agreement between the NE-8000 and the E-4000. In the differentiation of leukocytes, the NE-8000 and H-6000 showed good correlation and conformity for lymphocytes, neutrophils and eosinophils; for monocytes and basophils the conformity was poor. We also found a good correlation between the NE-8000 and E-4000 for lymphocytes and neutrophils, whereas the correlation was less for the middle cell ratio and the sum of monocytes, eosinophils and basophils. Furthermore, we compared Receiver Operating Characteristic curves for the different leukocyte subtypes on the NE-8000, the H-6000 and the E-4000, and investigated the flagging frequency in inpatients, outpatients and blood donors.

Blood Cell Count↗
PubMed2006

CD4+ T-cell counts, CD4+/CD8+ T-cell count ratios, and antibody levels in migrant fishermen infected with Schistosoma japonicum in the Dongting Lake, China.

In this study, CD4(+) and CD8(+) T cells and antibody levels were measured in 94 migrant fishermen infected with Schistosoma japonicum from Dongting Lake, China. Prevalence among these fishermen was high (63.8%), with a mean infection intensity of 61.4 +/- 3.8 epg, and included a high proportion of individuals (39.4%) with substantial parenchymal fibrosis (stages > or = 2/3). The CD4(+)/CD8(+) ratio in men (1.34 +/- 0.11) was significantly lower than that of women (1.58 +/- 0.15). CD4(+) T cells and the ratio of CD4(+)/CD8(+) were significantly decreased both in subjects infected with S. japonicum and those with parenchymal fibrosis. However, levels of total IgA, IgM, and anti-schistosome egg antigen IgG correlated positively with infection intensity and pathologic lesion number. These results suggest an imbalance between cell-mediated and humoral immunity in these fishermen, the precise cause of which remains undetermined.

Antibodies, Helminth↗
PubMed1994

Electronic cell counting to measure total cell numbers in bronchoalveolar lavage fluid.

Cell counting of bronchoalveolar lavage (BAL) fluid is performed manually in routine practice. This has both methodological and inherent errors; however, the accuracy and suitability of automated counting devices have been questioned. In this study, a Coulter Counter D Industrial model was calibrated and then used to measure the total cell count in unprocessed bronchoalveolar lavage fluid, and compared to a standard manual method. Bronchoalveolar lavage was performed on 34 patients undergoing routine bronchoscopy. An aliquot of unprocessed bronchoalveolar lavage fluid was taken for all counting procedures. Manual counts were performed blind by two experienced independent observers using improved Neubauer chambers. Electronic counting measured 1 ml aliquots suspended in 10 and 20 ml Isoton counting 0.5 and 1 ml duplicates. The correlation coefficients between electronic and manual counts were good. The coefficients of repeatability of electronic counts, for repeat counts, both on the same dilution (Intra-Coulter: 0.1 x 10(5) cells.ml-1) and different dilutions (Interdilution-Coulter: 0.48 x 10(5) cells.ml-1), were superior compared to those for repeat manual counts by the same observer (1.03 x 10(5) cells.ml-1), and counts between observers (1.82 x 10(5) cells.ml-1). This method offers a quick, precise and simple method for counting cells in unprocessed bronchoalveolar lavage fluid, which is both less labour intensive and subjective than manual counting.

Bronchoalveolar Lavage Fluid↗
PubMed1993

Software for counting cells and estimating structural volumes with the optical disector and fractionator.

We describe MS-DOS software for the optical volume fractionator (OVF), a stereological method combining the principles of the optical disector (Gundersen et al.: Acta Pathol. Microbiol. Immunol. Scand., 96:857-881, 1988) and fractionator (Gundersen: J. Microsc., 143:3-45, 1986). The OVF program estimates the volume of a fixed and embedded structure, the numerical density of cells, and the total number of cells in a structure. The hardware requirements include a PC computer (386 or 486 with VGA graphics) and a conventional light microscope fitted with a rotating stage, extension tube, and length gauge. The software includes an introduction, tutorial, simulator, laboratory tool kit, and report generator. The tool kit improves the efficiency of gathering stereological data with light microscopy and offers a convenient link between the data of light and electron microscopy. A novel algorithm, based on fractionator sampling, gives the volume of a fixed and embedded structure from the same set of sections used for cell counting. A laboratory example illustrates the operation of the software.

Algorithms↗
PubMed1998

Do viral load and CD8 cell count at initiation of tritherapy influence the increase of CD4 T-cell count?

BACKGROUND: Tritherapies including protease inhibitors improve clinical status and usually increase CD4 T cell count. However, the dissociation between the marked decreases in viral load and the incomplete restoration of CD4 cell counts with a three-drug combination has been reported. We assessed this potential difference among our patients. METHODS: Patients were enrolled when a protease inhibitor was prescribed to them for the first time. Using a computerized medical record (ADDIS), we retrospectively assessed a potential relationship between the increase in CD4 T cells (deltaCD4) at M3, M6 and variables including sex, age, CDC staging, protease inhibitor, prior antiviral therapy, CD8 and viral load at baseline. We used Epi-Info 6.4 and BMDP software. RESULTS: Data were analyzed on 154 patients. The median CD4 T cell count was 157 at baseline, 215 at month 3 and 202 at month 6. The median viral load was 52000 copies at baseline, 530 at month 3 and 500 at month 6. In a univariate analysis, a significant relationship was found between deltaCD4 and CD8 at baseline. A statistically significant negative correlation appeared between the CD8 cell count at baseline and deltaCD4 at M6 (r=-0.28, Pearson). Moreover, we found that there also was a relationship between deltaCD4 and viral load at baseline. There was a correlation between deltaCD4 at M6 and the viral load at M0 (r=0.37, Pearson). In a multiple regression model, after CD8 count at baseline had been accounted for, we found a significant correlation between deltaCD4 and viral load at baseline (multiple r=0.33 at M3, and 0.40 at M6). CONCLUSIONS: Patients with a low viral load do not benefit from as great an increase in CD4 T cell count as others when they receive a tritherapy including protease inhibitors. These results suggest that another mechanism rather than direct viral pathogenicity leads to CD4 T cell destruction. This mechanism may not be efficiently stopped by antiviral therapy, especially protease inhibitors.

Adult↗
PubMed1992

Correlation of histologic corneal endothelial cell counts with specular microscopic cell density.

The central endothelia of 48 eye bank corneas from donors ranging in age from 5 weeks to 88 years were photographed using in vitro specular microscopy. Computer-assisted morphometric analysis of the size and shape of endothelial cells was performed, and cell density was calculated. Histologic examination of the corneas after specular microscopy determined endothelial cell counts using x40 objective magnification. The mean endothelial cell counts from five different high-power fields were calculated. Results showed that there is a direct correlation between cell number and specular microscopy cell density (r = .91 and Spearman rank correlation, 0.69; both significant at P less than .01). A nomogram was developed to estimate corneal endothelial cell density from high-power field cell counts of pathologic specimens.

Adolescent↗
PubMed1976

Effects of thyroxine on T-cell counts and tumour cell rejection in mice.

In an attempt to study the effect of thyroxine on peripheral T-cell (thymus derived lymphocyte) counts or immunological functions, inbred C3H/He mice (8-10 weeks old) were injected subcutaneously with thyroxine for more than 3 months. After treatment for 3 months the mice were examined for peripheral T-cell counts, thymic incorporation of tritiated thymidine and rejection of tumour transplants. The number of T-cells was counted by the indirect immunoflourescence method using anti-thetaC3H serum after separation of lymphocytes on "Ficoll-Conray". It was revealed that the peripheral counts of both lymphocytes and T-cells were increased in the thyroxine treated group as compared with the control group, as was reported in the patients with Graves' disease. Thymic incorporation of tritiated thymidine was also found to be significantly increased in the thyroxine treated group. In addition, in order to study T-cell activity of the host, thyroxine treated and control mice were challenged with Ehrlich carcinoma cells at several concentrations (10(2), 10(4) and 2 x 10(6) per mouse). It was found that rejection of tumour transplants was significantly enhanced in the T-cell rich mice. Thus, it is possible that throxine affects peripheral T-cell counts and enhances immunological functions of the host.

Animals↗
PubMed2001

Use of monoclonal antibodies to refine flow cytometric differential cell counting of canine bone marrow cells.

OBJECTIVES: To evaluate use of monoclonal antibodies to increase accuracy of flow cytometric differential cell counting of canine bone marrow cells. SAMPLE POPULATION: Bone marrow specimens from 15 dogs. PROCEDURES: Specimens were labeled with monoclonal antibodies that detected CD18, major histocompatability antigen class-II (MHC class-II), CD14, and Thy-1. Location of fluorescent and nonfluorescent cells within gates of a template developed for canine bone marrow differential cell counting was determined, the template was revised, and 10 specimens were analyzed by use of the old and revised templates and by labeling cells with anti-MHC class-II and anti-CD14. RESULTS: Data confirmed the presumptive location of marrow subpopulations in scatter plots, permitted detection of lymphocytes and monocytemacrophages, and was used to revise the analysis template used for differential cell counting. When differential cells counts determined by the original and revised templates were compared with results of manual differential cell counts, the revised template had higher correlation coefficients and more similar mean values. Labeling cells with anti-MHC class-II and anti-CD14 permitted identification of lymphoid and monocyte-macrophages cells in bone marrow specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the revised flow cytometric analysis template combined with anti-CD14 and anti-MHC class-II antibody labeling provides reliable differential cell counts for clinical bone marrow specimens in dogs. These techniques have potential applications to clinical bone marrow examination and preclinical toxicity studies.

Animals↗
PubMed1997

Evaluation of low cost in-line milk samplers for estimating individual cow somatic cell counts.

The Dairy Herd Improvement Fund of Victoria recently identified a requirement for a simple and inexpensive in-line sampler to enable dairy farmers to collect representative milk samples for counting somatic cells. We found that the currently available simple in-line milk samplers, when connected to standard 35 ml collection vessels, terminate sampling early in a milking, and thus provide samples that are unrepresentative of the whole milking. We showed that cell count during a milking varies greatly, tending to be high for the first 1-21. Analyses of resulting samples will thus tend to overestimate cell counts if samplers are used in their traditional way. We found greater sampling rates in high-line compared with low-line milking systems, and consequently developed modified samplers suitable for both situations. Our samplers utilize low sampling rates (approximately 1-3%) and large collection vessels (450 ml). Compared with currently available simple in-line samplers, our type of sampler provided milk samples considerably more representative of the entire milking for the majority of cows. In conjunction with subsampling, they provided samples of appropriate size (12.5 ml minimum to 25 ml maximum) for testing fat, protein, lactose and cell count. Cell count results indicated that errors associated with the use of currently available simple in-line samplers could frequently be > 200%. In contrast, we found that use of our samplers gave an estimate for cell count that was only slightly higher (mean 20%) than that from samples collected by an approved Tru-Test sampler.

Animals↗
PubMedDate not supplied

Automated differential cell counting of viable cells.

A rapid, automated method of differentially counting viable cells by ethidium bromide-acridine orange cytofluorography is described (Takasugi 1971). The method is shown to have comparability, equal precision, and sensitivity to the standard trypan blue, visual method. The method is applicable to microcytotoxicity testing.

Automation↗
PubMed2006

On distinguishing cause and consequence: do high somatic cell counts lead to lower milk yield or does high milk yield lead to lower somatic cell count?

Researchers have reported that as milk yield increases composite milk somatic cell count (SCC) is diluted in cattle with no intramammary infection (IMI) and as a consequence, estimates of SCC from high yields are lower than estimates of SCC from low yields in dairy cows without an IMI. To date, estimates of reduced milk yield associated with high SCC because of intramammary infection have not been adjusted for any dilution of SCC. Ignoring dilution is therefore likely to lead to an overestimate of reduction in yield with increasing SCC. This paper investigates scenarios of the possible impact of dilution and inflammation on the association between somatic cell count and yield. The data used to investigate this relationship come from 8373 monthly records of milk yield and composite somatic cell count, together with incidence of clinical mastitis, which were recorded on 850 cows from five dairy cattle farms in Gloucestershire, UK. Two sets of models were used to investigate dilution and inflammation using two-level hierarchical models. The first set of models was used to estimate the linear (dilution) and log10-linear (inflammation) impact of SCC on the outcome variable milk yield. Five general linear models with increasing inclusion of higher test day SCC values were run. The cumulative categories were test day SCC values of up to and inclusive of 30, 50, 100, 200 and 400x10(3)cells/ml. Linear and log linear SCC influences on milk yield were estimated. At low SCC values the linear SCC predictor was dominant, while at higher values the log linear predictor was dominant. Up to 100x10(3)cells/ml there was mostly a slightly negative linear relationship between SCC and yield, potentially indicating a dilution effect. In the second set of models, three approaches to adjust milk loss for dilution were compared with an unadjusted model. In general, dilution-adjusted SCC values fitted the data better and resulted in a slightly lower milk loss per SCC category compared with unadjusted SCC. In all models with a dilution term there was a significant reduction in yield with SCC>200x10(3)cells/ml.

Animals↗
PubMed2001

Usefulness of route of transmission, absolute CD8+ T-cell counts, and levels of serum tumor necrosis factor alpha as predictors of survival of HIV-1-infected patients with very low CD4 + T-cell counts.

Potential cofactors of survival in HIV-1-infected patients with CD4+ T-cell counts of < or = 100 cells/microl were investigated. All 132 patients with CD4+ T-cell counts of < or = 100 cells/microl were selected from 416 patients included in an antiretroviral therapy cohort (1989-1999). Fifty of 54 deaths were due to AIDS. There were significant associations (P<0.05) between survival and CD8+ T-cell counts, clinical AIDS stage, risk group, and antiretroviral drug regimen after baseline, but only the use of protease inhibitors had an independent effect on survival (hazard ratio [HR], 0.096; 95% confidence interval [95%CI], 0.094-0.097). A substudy restricted to the cohort of 108 patients never exposed to PIs detected independent associations between survival and CD8+ T-cell counts (P=0.0016), experience with antiretroviral therapy before baseline (HR, 2.52; 95%CI, 1.31-4.82), sexual risk group for HIV infection (HR, 3.7; 95%CI, 1.92-7.12), and levels of serum tumor necrosis factor alpha (P=0.02). This study confirms that the use of PI-containing antiretroviral regimens strongly predicts survival of HIV-1-infected patients with very low CD4+ T-cell counts. When the study was restricted to patients never exposed to PIs, the parenteral route of disease transmission, high absolute CD8 + T-cell counts, and low serum levels of tumor necrosis factor alpha were independent predictors of survival in extremely advanced HIV-1 disease.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Pyramidal neuron size in the hippocampus of schizophrenics correlates with total cell count and degree of cell disarray.

Hippocampal pyramidal neuron size was determined in all Cornu Ammonis subregions - CA1-CA4 - in five chronic schizophrenic men and compared with eight controls matched with respect to age and sex. Four out of five probands and the same eight controls had been examined in a previous study showing a significantly lower cell count and disorientation of pyramidal cells in the CA1- CA3 subregions of the schizophrenics. There was also a negative correlation between the total number of cells and the number of disoriented cells. In this study it was shown that the schizophrenic probands also had significantly smaller neurons in all subregions. There was a significant negative correlation between pyramidal neuron size and the number of disarrayed neurons in each subregion, and there was a significant positive correlation between neuron size and the total number of pyramidal cells in CA1 and CA2, but not in CA3 and CA4. The consistency of hippocampal anomalies in these schizophrenics is, thus, demonstrated by the statistical relations between the different parameters examined.

Age Factors↗
PubMed1993

Histologic grading in soft-tissue sarcomas. An analysis of 194 cases including AgNOR count and mast-cell count.

In order to establish a new histologic grading system for STS, we evaluated histologic prognostic factors. For this purpose, we selected 194 patients with STS: 31 in the upper extremities, 63 in the trunk, and 100 in lower extremities. All the patients were treated by surgery, followed by chemotherapy in 74 cases, radiotherapy in 11, chemotherapy and radiotherapy in 30, or no adjuvant treatment in 79. Histologic factors evaluated were mitotic count, extent of necrosis, cellularity, cellular pleomorphism, extent of myxoid change, sclerosis, non-specific histologic diagnosis, counting of reaction product in silver stain for nucleolar organizer regions (AgNOR) and mast-cell counts. Univariate analysis revealed mitotic count, necrosis, cellularity, cellular pleomorphism, non-specific histologic classification, AgNOR count and mast-cell count to be significantly related to prognosis. Multivariate analysis revealed that AgNOR count, cellularity and necrosis were independent prognostic factors. A new grading system was introduced: low-grade, intermediate-grade and high-grade. The survival between each group were significantly different; the 5-year-survival rate in patients of the low-, intermediate- and high-grade groups was 87%, 74% and 35% respectively. Our findings suggest that this histologic grading system may be useful for making therapeutic decisions.

Adolescent↗