Estimation of the number of stellate cells in a liver with the smooth fractionator.
To 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 congruent with 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 ( congruent with 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.
Marcos
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Laboratory of Histology and Embryology, Institute of Biomedical Sciences Abel Salazar, ICBAS, University of Porto, Portugal.
Monteiro
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Rocha
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D000818:Animals; D002469:Cell Separation / Q000379:methods; D022781:Hepatocytes / Q000166:cytology / Q000473:pathology; D008099:Liver / Q000166:cytology* / Q000473:pathology*; D008103:Liver Cirrhosis / Q000473:pathology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012984:Software