Design-based stereological estimation of hepatocyte number, by combining the smooth optical fractionator and immunocytochemistry with anti-carcinoembryonic antigen polyclonal antibodies.
OBJECTIVE
Hepatocytes (HEP) have been the major target for structural quantification in the liver, but an estimation of their total number (N), their percentage in relation to the global number of liver cells and the evaluation of the percentage of binucleated hepatocytes (BnHEPs) have never been performed with modern design-based stereological techniques. The establishment of sound technical guidelines and baseline quantitative data in non-pathological conditions are relevant to properly evaluate HEP hyperplasia and BnHEP responses.


METHODS
In this study, we combined immunocytochemistry with sound design-based stereology for estimating the N of HEP and the N of non-hepatocytic cells (NHCs). For obtaining systematic uniform random sections (30 microm thick), a smooth fractionator sampling scheme was applied to the liver of five male Wistar rats (3 month old). Those sections were immunostained with polyclonal antibodies against carcinoembryonic antigen. Because biliary canaliculi were then marked, an unequivocal counting of mononucleated hepatocytes (MnHEP) and BnHEP was allowed.


RESULTS
The N of HEP was estimated to be 1.93 x 10(9), with a coefficient of error (CE) of 0.02, corresponding to 129 x 10(6) HEP/g of liver. BnHEP represented 26% of total HEP number. The N of NHC was estimated as 1.31 x 10(9) (CE=0.02).


CONCLUSIONS
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.
Marcos
Ricardo
R
Laboratory of Histology and Embryology, ICBAS - Institute of Biomedical Sciences Abel Salazar, University of Porto - UP, Portugal. rmarcos@icbas.up.pt
Monteiro
Rogério A F
RA
Rocha
Eduardo
E
D000818:Animals; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D004195:Disease Models, Animal; D022781:Hepatocytes / Q000166:cytology* / Q000276:immunology*; D006652:Histological Techniques; D007150:Immunohistochemistry; D008103:Liver Cirrhosis / Q000473:pathology*; D008297:Male; D011336:Probability; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric
D002272:Carcinoembryonic Antigen