Stereology offers a number of tools for the analysis of sections in microscopy (which usually provide only twodimensional information) for the purpose of estimating geometric quantities, such as volume, surface area, length or number of particles (cells or other structures). The use of these tools enables recovery of the threedimensional information that is inherent in biological tissues. This review uses the liver as a paradigm for summarizing the most commonly used state-of-the-art methods for quantitation in design-based stereology. Because it is often relevant to distinguish hyperplasia and hypertrophy in liver responses, we also focus on potential pitfalls in the sampling and processing of liver specimens for stereological purposes, and assess the existing methods for volume and number estimation. With respect to volume, we considered whole liver volume (V), volume density (V V ) and so-called local volumes, including the number-weighted volume ( v N ) and the volume-weighted volume ( v v ). For number, we considered the total number (N) and the numerical density (N V ). If correctly applied, current stereological methods guarantee that no bias is introduced in the estimates, which will be therefore accurate; additionally, methods can be tuned for obtaining precise quantitative estimates that can reveal subtle changes in the volume or number of selected hepatic cells. These methods have already detailed the effects of some substances and specific diets on the liver, and should be routinely included in the toolbox of liver research.
A 4-year-old, domestic shorthair, female spayed cat was presented for decreased appetite and depression. Severe pancytopenia with erythrocyte autoagglutination was found. The cat was seronegative for feline immunodeficiency and leukemia viruses. Immune-mediated hemolytic anemia was suspected but no response to treatment with a blood transfusion, enrofloxacin, and prednisone was observed. Blood and bone marrow smears obtained 11 days later contained Leishmania amastigotes in the cytoplasm of neutrophils and macrophages, respectively. Serologic and PCR testing of peripheral blood confirmed infection with Leishmania infantum. Despite treatment, the cat worsened clinically and was euthanized. At necropsy, visceral dissemination of the parasite was confirmed. The findings in this case indicate that visceral leishmaniasis should be considered as a differential diagnoses in cats with pancytopenia in areas endemic for Leishmania. In addition, amastigotes may be observed in peripheral blood neutrophils.
The CTB method was easily implementable under practice conditions (up to the fixation of the microhemtocrit tube), analogous to surgical biopsy submission for histology. Cell tube blocks can increase diagnostic accuracy when the technique is used in tandem after the cytologic evaluation, and the technique allows storage of fluids. Other advantages of CTB were the simplicity, low cost, and separation of erythrocytes from the nucleated cells, which was helpful in hemodiluted samples.
The strategy here presented provides a reliable method for accessing the N of HEP (distinguishing MnHEP from BnHEP) in situations in which these parameters are relevant, namely for evaluating the magnitude of an hyperplastic liver response from its very early onset.
SummaryTo better evaluate the activation and proliferative response of hepatic stellate cells (HSC) in hepatic fibrosis, it is essential to have sound quantitative data in non-pathological conditions. Our aim was to obtain the first precise and unbiased estimate of the total number of HSC in the adult rat, by combining the optical fractionator, in a smooth sampling design, with immunocytochemistry against glial fibrillary acidic protein.Moreover, we wanted to verify whether there was sufficiently relevant specimen inhomogeneity that could jeopardize the high expected estimate precision when using the smooth fractionator design for HSC. Finally, we wanted to address the question of what sampling scheme would be advisable a priori for future studies. Microscopical observations and quantitative data provided no evidence for inhomogeneity of tissue distribution of HSC. Under this scenario, we implemented a baseline sampling strategy estimating the number ( N ) of HSC as 207E 06 (CV = 0.17). The coefficient of error [CE( N )] was 0.04, as calculated by two formerly proposed approaches. The biological difference among animals contributed ≅ 95% to the observed variability, whereas methodological variance comprised the remaining 5%. We then carried out a half reduction of sampling effort, at the level of both sections and fields. In either occasion, the CE( N ) values were low ( ≅ 0.05) and the biological variance continued to be far more important than methodological variance. We concluded that our baseline sampling (counting 650 -1000 cells/rat) would be appropriate to assess the lobular distribution and the N of HSC. However, if the latter is the only parameter to be estimated, around half of our baseline sampling (counting 250-600 cells/rat) would still generate precise estimates [CE( N ) < 0.1], being in this case more efficient to reduce the number of sections than to reduce the sampled fields.
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