Influence of fixation and immunohistological technique on accuracy, precision and inter-observer reproducibility of plasma cell counting.

Recently two highly sensitive and specific diagnostic criteria for Sj?gren's syndrome based on percentages of IgA-, IgG-, and IgM-containing plasma cells measured in immunohistologically stained labial salivary gland tissue have been described. The reliability of such a criterion is dependent on the accuracy, precision and inter-observer reproducibility in plasma cell counting. The present study evaluates the effect of tissue fixation and immunohistological procedures on the aforementioned factors. Immunoglobulin (Ig)-containing plasma cells in sections of lamellated submandibular salivary gland tissue, alternately fixed in a 4% buffered formol solution or formol-sublimate solution and stained with an indirect immunoperoxidase and unlabelled peroxidase anti-peroxidase (PAP) method respectively, were enumerated by three independent observers. Relative numbers of Ig-containing plasma cells appeared to be less sensitive for systematic errors due to tissue fixation and immunohistological procedure than absolute numbers of Ig-containing plasma cells. The best inter-observer reproducibility of plasma cell counts was obtained in sections from formol sublimate-fixed specimens stained according to the PAP procedure.     

An Ocular Grid-Planimeter Method for Enumerating Nerve Fibers

The estimation of numbers of nerve fibers in cross sections of peripheral nerves containing both fine and large fiber components can be accomplished by using an ocular grid and selectively counting a known area. With the use of a projecting apparatus and planimeter, the total cross section area is determined.

The following proportion expresses the principle involved:

total number of fibers in the nerve area of ass section of entire nerve number counted in the sample area area of sample

I f the planimeter calibration and the magnification of the tracing remain the same, a constant factor may be used for successive estimates. This factor is equal to the value of the planimeter reading of 1.000 divided by the area of the grid times the magnification squared. The final calculations are made by multiplying the number of fibers counted times the planimeter reading times the constant and dividing by the number of grid squares counted.

Counts of some nerves, using high magnification in enumerating the sample areas, can be finished in less than an hour after the preparation of slides. In comparing numbers obtained by complete counts with estimated numbers, the error was determined to be approximately ± 5%     

Comparative study of the abundance of various bacterial morphotypes in an eutrophic freshwater environment determined by AODC and TEM

Transmission electron microscopy (TEM) and epifluorescence microscopy were used to obtain comparative measurements of total bacterial counts, and to enumerate abundances of various bacterial morphotypes in an eutrophic freshwater habitat. Although particulate matter would have been expected to interfere with counting by obscuring large areas of the electron microscope grids, estimates of total bacterial abundance made by TEM were on average 1.2 times greater than those obtained using the acridine orange direct counting method (AODC). However, the precision of the AODC method was greater than that for TEM, with a coefficient of variation (C.V.) of 4.0% versus 8.8%, respectively. The total bacterial abundance ranged from 1.1 to 3.2 x 10(6) ml(-1). As was the case for total bacterial density, the numbers of rod- and vibrio-shaped cells were lower when counted in the epifluorescence microscope, indicating the presence of potential starvation forms or ultramicrobacteria. Greatest variations in counts made by TEM and AODC were found for filamentous and coccoid bacteria. Counts of filamentous bacteria made by AODC were only about half of those detected by TEM. In contrast, cocci were on average 1.5 times greater when counted by AODC compared to TEM estimates. Both counting differences were probably caused by the morphology and low density of filamentous and coccoid bacteria (1.7 and 1.4 x 10(5) ml(-1), respectively), which led to an uneven distribution on polycarbonate filters as well as on electron microscope grids. Besides, cocci might easily be mistaken for large viral particles when counted by AODC. Hence, the study supports the use of TEM over AODC for obtaining accurate estimates of total bacterial abundance and especially bacterial morphotypes in natural waters.     

Optimization of direct cell counting in sediment

This study reports a method for optimizing direct counts of bacteria in sediment, designed to reduce the masking by sediment particles. The protocol was designed to determine appropriate dilution factors by incorporating counting statistics and was used to measure depth-associated changes in microbial abundance in metal-impacted freshwater sediments. We demonstrated a direct method to determine appropriate sample dilution for accurate counting by adding a known amount of cells to the sediment. For accurate counting in our sediment samples, we determined that the average number of bacteria per microscope ocular field must be between 8.5 and 10. This is well below the 30 bacteria/field previously suggested for accurate counting. These results indicate that an optimal dilution rate must be determined before accurate direct counts in sediment can be achieved.     

Inducible nitric oxide synthase activity in colon biopsies from inflammatory areas: correlation with inflammation intensity in patients with ulcerative colitis but not with Crohn's disease

Summary. Nitric oxide synthase (NOS) activities are responsible for the enzymatic conversion of L-arginine into NO and L-citrulline. Relatively low amounts of NO are produced in intestinal epithelial cells or are released from nerve endings. The effects of NO production are related to the maintenance of epithelial integrity and permeability. A pathological role of an increased NO production has been suggested to play a role in models of experimental colitis. In humans, NOS activity in colon mucosa from patients with ulcerative colitis is clearly increased when compared with the activity of the control group. In contrast, an increase of NOS activity in the colon mucosa from patients with Crohn's disease remains controversial. In the present work, we have measured NOS activity in colon biopsies originating from the control group (n = 16), from patients with ulcerative colitis (n = 23) and Crohn's disease (n = 17) using the radiochemical method of the conversion of L-[guanido-¹⁴C] arginine into radioactive L-citrulline. In the control group, NOS activity was mainly of the inducible type (88% of total NOS activity) since it was characterised by its insensibility to the absence of calcium in the assay medium. In colon biopsies originating from patients with ulcerative colitis, inducible NOS activity was increased 3 fold (p < 0.005) and in patients with Crohn's disease, inducible NOS activity was increased 5 fold (p < 0.005). Correlations between NOS activity in colon biopsies and the intensity parameters of the disease i.e. Truelove index, endoscopic score and histo-logical parameters were evidenced in patients with ulcerative colitis. In contrast, in patients with Crohn's disease, the high inducible NOS activity was not correlated with any intensity parameters of the disease. From these data, we concluded that although inducible NOS activity was increased several fold in colon biopsies originating from patients with both ulcerative colitis and Crohn's disease, a correlation between this activity and the severity of bowel inflammation was not found in either cases. Received August 7, 1999     

A method for the quantification ofFrankia by estimation of hyphal length

Summary The cellular mass ofFrankia, a filamentous actinomycete, was readily quantified by estimating hyphal length, using a modification of Tennant's method for the estimation of root length. Each sample ofFrankia was stained with Coomassie Brilliant blue G 250, dispersed well, and suspended in a 0.5% agar solution. One drop of the suspension was placed in a Petroff-Hausser counting chamber with 0.05 by 0.05mm grid squares. The number of intersections betweenFrankia hyphae and the grid lines in a standard area were counted under a microscope and converted to hyphal length. Using the formula: hyphal length (HL) in mm equals (11/14) times the number of intersections (n) times the grid dimension (0.05 mm). The validity of the line intersection method was tested by comparison with total protein estimates of replicate aliquots ofFrankia culture. Correlations between total protein and hyphal length estimates were strong (r² from 0.76 to 0.95; standard errors of 3 to 9% of estimated length). These results show that line intersection counts may be a satisfactory routine method for quantifyingFrankia in culture and may be especially suitable for detecting small amounts of livingFrankia in less time than with other methods.Dedicated to the memory of Professor John G. Torrey     

Flow-cytometric cell counting and DNA estimation for the study of plant cell population dynamics

A one-step procedure is presented for simultaneous measurement of cell number and DNA content in cultured plant cells by flow cytometry. In order to obtain nuclei representative of the growth stadium of the culture and of all phases of the cell cycle, cells were carefully sampled and immediately fixed. Next, nuclei were isolated by enzymatic and mechanical maceration, and stained with a DNA-specific fluorescent dye. In the resultant preparation, cells can be counted at relative ease by means of a fluorescence microscope. However, flow-cytometric counting appeared to be superior to manual counting since the time needed for flow-cytometric counting was one-fourth that for manual counting and the variance between counts of the samples was significantly less. In addition, from the same routine, accurate DNA distributions were obtained as a second important parameter of the population dynamics.     

Flow cytometric-based isolation of nucleated erythroid cells during maturation: an approach to cell surface antigen studies

Nucleated red blood cells (NRBCs) are involved in normal physiologic processes, as well as in several malignancies. They are usually counted manually under the microscope. However, blood sample manipulation may be a source of variability and manual counting is imprecise, time-consuming, and subjective. To improve identification of CD45-negative cells, we used a flow cytometry technique that avoids the addition of lysing reagents and stains viable cell nuclei. We applied this method for counting and isolating NRBC subpopulations in whole blood samples, using DNA/RNA viable staining to discriminate nonnucleated erythroid cells and debris. NRBC counts gave 197.95 cells per mm(3) in mobilized peripheral blood samples (1.00%, n = 20), 3897.59 cells per mm(3) in leukapheresis products (3.08%, n = 20), and 765.21 cells per mm(3) in cord blood samples (6.09%, n = 20). Normal bone marrow counts were 5449.42 cells per mm(3) (11.76%, n = 20). Scatter profiles showed three distinct populations, from early to late-stage erythroblasts, consisting of erythroblasts, orthochromatic erythroblasts, and ejected nuclei, as confirmed by Wright-Giemsa staining. In addition, flow cytometry immunophenotyping showed that glycophorin A was expressed dimly on NRBCs during maturation. These findings point to the feasibility of live NRBCs studies, which offer great potential for a wide range of disciplines.     

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed¹ who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate².To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry¹. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments¹.The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection³ in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.     

The genetics and immunopathogenesis of inflammatory bowel disease

Genome-wide association studies efficiently and powerfully assay common genetic variation. The application of these studies to Crohn's disease has provided insight into the immunopathogenesis of this disease, implicating a role for genes of the innate and adaptive immune systems. In this Review, I discuss our current understanding of the genetics and immunopathogenesis of Crohn's disease and ulcerative colitis. Crohn's disease, but not ulcerative colitis, is associated with genetic variation in NOD2 and an autophagy gene, ATG16L1, both of which affect the intracellular processing of bacterial components. By contrast, variation in the gene encoding the interleukin-23 (IL-23) receptor subunit, as well as in the IL12B, STAT3 and NKX2-3 gene regions, is associated with both Crohn's disease and ulcerative colitis. Comparative analyses of gene associations between these two inflammatory bowel diseases reveal common and unique mechanisms of their immunopathogenesis.     

The role of activated cytotoxic T cells in inflammatory bowel disease

The role of cell-mediated cytotoxicity in the pathogenesis of ulcerative colitis and Crohn's disease has been controversial since reports indicating either a decreased, or an increased, activity of cytotoxic T cells in active stages of inflammatory bowel disease exist. Some of these discrepancies may be attributed to the fact that so far mostly peripheral blood lymphocytes rather than intestinal T cells have been examined. To overcome some of these limitations we performed in situ hybridizations for the detection of perforin and granzyme A mRNA expressing cells, i.e. of cytotoxic cells activated in situ, in the affected intestinal mucosa. These studies revealed increased frequencies of activated, cytotoxic T cells in active stages of ulcerative colitis and Crohn's disease. Interestingly, activated perforin mRNA expressing T cells are present both in the CD4 and in the CD8 T cell subsets. In the latter T cell subset up to 60% of the mucosal T cells isolated from the affected sites express perforin mRNA at detectable levels. The elevated frequency of activated cytotoxic cells and their histological distribution also in close proximity to the epithelial cells may thus indicate an important role for cytotoxic cells in the pathogenesis of inflammatory bowel disease since activated cytotoxic T cells may further exacerbate the inflammatory process through the production of pro-inflammatory cytokines such as interferon-gamma or tumor necrosis factor-alpha, but also through the release of pro-inflammatory cytokines and chemokines upon lysis of epithelial cells and the increased influx of luminal antigens at the site of epithelial erosions.     

Degree of variation and reproducibility of different methods for the diagnosis of subclinical endometritis

Endometrial cytology as a reliable diagnostic technique has been established for the diagnosis of subclinical endometritis (SE) in cows. Several counting techniques have been used to determine polymorphonuclear neutrophils (PMN) in endometrial samples. Information on the agreement between different techniques, however, is limited. Therefore, the objective of this study was to analyze the degree of variation in the percentage of endometrial cells and PMN determined by six different counting techniques. A second objective was to evaluate the interobserver reproducibility of the cell counting by two different examiners. One hundred samples were examined by the different counting techniques. The applied methods counted a total of 100, 300, or 500 cells (C100, C300, C500), respectively. In addition, method HPF100 and HPF300 counted 100 and 300 cells in 10 high-power fields per slide. Finally, one method estimated (EST) the percentage of PMN by screening the slide under the microscope. The interobserver reproducibility between two examiners was analyzed for method C300. The comparison between the six different methods showed a strong compliance (r = 0.77–0.90) with greatest correlation coefficient between C100 and C300. The results of Kappa statistics revealed agreement between methods varying from ? = 0.30–0.85, with the greatest agreement between HPF300 and EST. Furthermore, the impact of the different methods on the resulting prevalence of SE was calculated, with the greatest prevalence determined by C100 (33.0%) and the least by HPF300 (10.0%). The results of the interobserver reproducibility showed good correlation and agreement (r = 0.86, ? = 0.79). In conclusion, all examined methods were suitable for the cytological evaluation of PMN, with method C100 showing lowest agreement with the other methods. This confirms the hypothesis that a suitable threshold for PMN is not only influenced by, for example, time of sampling postpartum, but also by the diagnostic method. A threshold of 5% PMN seems to be useful when C300 and HPF100 are used, whereas counting 100 cells or estimating the percentage of PMN seems to overestimate or underestimate the prevalence of SE, respectively. In conclusion, method C300 and HPF100 can be recommended as methods of choice for evaluating the percentage of PMN in endometrial samples to diagnose SE.     

Improved Aerobic Colony Count Technique for Hydrophobic Grid Membrane Filters

The AOAC International official action procedure for performing aerobic colony counts on hydrophobic grid membrane filters (HGMFs) uses Trypticase soy-fast green FCF agar (FGA) incubated for 48 h. Microbial growths are various shades of green on a pale green background, which can cause problems for automated as well as manual counting. HGMFs which had been incubated 24 or 48 h at 35°C on Trypticase soy agar were flooded underneath with 1 to 2 ml of 0.1% triphenyltetrazolium chloride (TTC) solution by simply lifting one corner of the filter while it was still on the agar and adding the reagent. Microbial growths on HGMFs were counted after color had been allowed to develop for 15 min at room temperature. With representative foods, virtually all colonies stained pink to red. Automated electronic counts made by using the MI-100 HGMF Interpreter were easier and more reliable than control HGMF counts made by the AOAC International official action procedure. Manual counting was easier as well because of increased visibility of the microbial growths. Except in the case of dairy products, 24-h TTC counts did not differ significantly from 48-h FGA counts, whereas the FGA counts at 24 h were always significantly lower, indicating that for many food products the HGMF TTC flooding method permits aerobic colony counts to be made after 24 h.     

Significant increase of interleukin 6 production in blood mononuclear leukocytes obtained from patients with active inflammatory bowel disease

In the present study, we compared the potency of interleukin 6 production in peripheral blood mononuclear leukocytes between paired patients with active stage and inactive stage of inflammatory bowel disease. Subjects included nine patients with ulcerative colitis, ten patients with Crohn's disease and sex-matched nine healthy volunteers. Mononuclear leukocytes were stimulated with concanavalin A for 24 h to induce interleukin 6 production. Interleukin 6 content in the culture medium was assayed by using specific ELISA and interleukin 6 dependent cell line MH-60. Interleukin 6 production was found to be significantly increased in mononuclear leukocytes from both active ulcerative colitis and Crohn's disease as compared to that from control subjects. There was no significant difference in interleukin 6 production between ulcerative colitis and Crohn's disease. The potency of interleukin 6 production was returned to the control level when the diseases became inactive. The present results, therefore, may indicate some important role of interleukin 6 in the pathogenesis of inflammatory bowel disease and also the potency of interleukin 6 production in mononuclear leukocytes can be an indicator of the activity of inflammatory bowel disease.     

Ultrastructural study of inflammatory bowel disease.

Ultrastructural changes that occurred in chronic active ulcerative colitis and Crohn's disease were investigated and compared to normal as well as to higher grades of dysplasia in adenomas and carcinomas. A greater number of immature absorptive cells, undifferentiated and intermediate cells were seen as compared to normal. One case of Crohn's and two cases of chronic ulcerative colitis including one with coexisting carcinoma showed increased number of vesicles and electron-dense bodies (EDB) in the absorptive cells and increased heterogeneity of mucin droplets in goblet cells and presence of atypical secretory cells (ASC). Higher grades of dysplasia characterised by large numbers of atypical secretory cells were not seen in the present series and provide no relationship between the atypical ultrastructural features and increased risk of malignancy. However, the number of cases investigated is too small and a large series is required to clarify the significance of observations such as increased number of electron-dense bodies and vesicles in the apical cytoplasm and presence of atypical secretory cells.     

Apoptosis: one of the mechanisms that maintains unresponsiveness of the intestinal mucosal immune system

Intestinal mucosa is constantly exposed to environmental AGS: Activation of lamina propria (LP) T cells by luminal Ags may lead to the production of inflammatory cytokines and subsequent mucosal inflammation and tissue damage. However, in normal circumstances, LP T cells do not respond to antigenic stimulation. The mechanisms of this unresponsiveness in healthy subjects are not fully understood. In this study, we found by in vivo analysis that, except for T cells in lymph nodules of the mucosa, 15% of LP T cells underwent apoptosis in normal individuals. In contrast, there was a marked reduction in apoptosis of LP T cells in patients with inflammatory bowel disease (Crohn's disease and ulcerative colitis) and those with specific colitis. Our findings suggest that apoptosis might be a mechanism that turns off mucosal T cell responses to environmental Ags in healthy subjects, and resistance to apoptosis could be an important cause of mucosal immune dysregulation and tissue inflammation in colitis.     

Pollen and fungal spore sampling and analysis. Statistical evaluations

Statistical evaluations of samples obtained from a Burkard seven-day recording volumetric pollen/spore trap were performed to determine the precision of the sampling and analysis procedures. The reproducibility of co-located traps was also investigated. The results showed that pollen grain transect counting was not significantly different, while fungal spore counting produced statistically different results. There was no statistical difference in the number of pollen and fungal spores counted between the co-located samplers. Reasons for the differences in the fungal spore counts are presented.     

ICMSF methods study. XVII. An international comparative study of the direct plate and hydrophobic grid-membrane filter methods for enumeration of Escherichia coli in foods. International Commission on Microbiological Specifications for Foods

Eight laboratories compared counts of Escherichia coli from naturally or artificially contaminated ground beef, other meats and poultry, vegetables, fish and shellfish, cheese, and diverse sources such as swabs, by the Anderson-Baird-Parker direct plate (DP) and a hydrophobic grid-membrane filter (HGMF) method. For five of the eight laboratories overall counts by HGMF were significantly low (51-83%) compared with those by DP. Counts by HGMF tended to be lower for naturally contaminated samples; several possible causes were investigated. In a subsidiary study, analyst variation in counting HGMF ranged from 0.8-7.3%, with little evidence of effects from counting positive versus negative grid cells or from the fullness of growth or staining intensity.     

Adhesive Escherichia coli in inflammatory bowel disease and infective diarrhoea.

The clinical features of ulcerative colitis and Crohn''s disease are similar to those of infections of the bowel, although their cause is uncertain. Many bacteria that cause intestinal diseases adhere to the gut mucosa, and adhesion of pathogenic Escherichia coli is resistant to D-mannose. The adhesive properties of isolates of E coli were assessed by assay of adhesion to buccal epithelial cells with mannose added. The isolates were obtained from patients with inflammatory bowel diseases (50 with a relapse of ulcerative colitis, nine with ulcerative colitis in remission, 13 with Crohn''s disease, and 11 with infectious diarrhoea not due to E coli) and 22 controls. The median index of adhesion to buccal epithelial cells (the proportion of cells with more than 50 adherent bacteria) for E coli from patients with ulcerative colitis in relapse was significantly higher (43%) than that for controls (5%) and patients with infectious diarrhoea (14%). The index was not significantly different among isolates from patients with ulcerative colitis in relapse, Crohn''s disease (53%), and ulcerative colitis in remission (30%). If an index of adhesion of greater than 25% is taken as indicating an adhesive strain 86% of isolates of E coli from patients with inflammatory bowel disease were adhesive compared with 27% from patients with infective diarrhoea and none from controls. The adhesive properties of the isolates from patients with inflammatory bowel disease were similar to those of pathogenic intestinal E coli, raising the possibility that they may have a role in the pathogenesis of the condition; the smaller proportion of adhesive isolates in patients with infective diarrhoea due to other bacteria suggests that the organism may be of primary importance rather than arising secondarily.     

Association of genetic variants of mannan-binding (MBL) lectin-2 gene, MBL levels and function in ulcerative colitis and Crohn's disease

Ulcerative colitis and Crohn's disease are the two major forms of inflammatory bowel disease (IBD). A series of reports have hypothesized interplay of genetic and environmental factors in the pathogenesis of IBD. Polymorphism in the mannan-binding lectin-2 (MBL-2) gene is known to affect the structural assembly and function thereby predisposing subjects to various diseases. The present study was designed to evaluate effect of MBL-2 gene polymorphism on MBL levels and function in IBD patients. Genomic DNA was isolated from blood samples collected from 157 ulcerative colitis, 42 Crohn's disease and 204 control subjects. Genotyping for different polymorphic sites at exon1 of MBL-2 gene was performed by refractory mutation system-PCR and amplification followed by restriction digestion (PCR-RFLP). Serum MBL concentration and C4 deposition levels were estimated using ELISA. Mannan-binding lectin-2 genotypic variants were calculated in IBD and healthy controls. The frequency of single nucleotide polymorphisms at codon 54 was significantly higher in ulcerative colitis patients than controls (P?相似文献