Skip to main content

Advertisement

SpringerLink
Log in

A critical analysis of three quantitative methods of assessment of hepatic steatosis in liver biopsies

  • Original Article
  • Published:
Virchows Archiv Aims and scope Submit manuscript

Abstract

The issue of adequately quantitatively evaluating hepatic steatosis is still unresolved. Therefore, we compared three methods of quantitative assessment. Two groups of mice (n = 10 each) were fed standard chow (10% fat, SC group) or a high-fat diet (60% fat, HF group) for 16 weeks, and hepatic triglyceride (HT) and liver tissue were then studied. Paraplast-embedded tissues stained by hematoxylin and eosin (H-E) were compared to frozen sections stained by Oil Red-O (ORO). In addition, the volume density of steatosis (Vv[steatosis, liver]) was measured by point counting (P-C, sections H-E or ORO) or by image analysis (I-A, sections ORO). HT was significantly higher in the HF group (104% greater, P = 0.0004) than in the SC group. With P-C and H-E, Vv[steatosis, liver] was 4.80 ± 0.90% in the SC group and 33.50 ± 3.17% in the HF group (600% greater, P < 0.0001). With P-C and ORO, Vv[steatosis, liver] was 4.86 ± 0.89% in the SC group and 25.21 ± 1.27% in the HF group (420% greater, P < 0.0001). With I-A and ORO, Vv[steatosis, liver] was 4.17 ± 0.85% in the SC group and 23.35 ± 1.58% in the HF group (460% greater, P < 0.0001). Correlations between Vv[steatosis, liver] and HT were strong and significant in all methods. In conclusion, all methods were appropriate and reproducible. In P-C and H-E, there is a slight overestimation of steatosis in the HF animals in comparison to frozen sections and ORO; in frozen sections, differences between P-C and I-A are insignificant.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Erickson SK (2009) Nonalcoholic fatty liver disease. J Lipid Res 50(Suppl):S412–416

    Article  PubMed  Google Scholar 

  2. Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy SM, Hobbs HH (2004) Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 40:1387–1395

    Article  PubMed  Google Scholar 

  3. Farrell GC, Larter CZ (2006) Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 43:S99–S112

    Article  PubMed  CAS  Google Scholar 

  4. Brunt EM, Tiniakos DG (2010) Histopathology of nonalcoholic fatty liver disease. World J Gastroenterol 16:5286–5296

    Article  PubMed  Google Scholar 

  5. Burt AD, Mutton A, Day CP (1998) Diagnosis and interpretation of steatosis and steatohepatitis. Semin Diagn Pathol 15:246–258

    PubMed  CAS  Google Scholar 

  6. Sherlock S (1995) Alcoholic liver disease. Lancet 345:227–229

    Article  PubMed  CAS  Google Scholar 

  7. Tannapfel A, Denk H, Dienes H-P, Langner C, Schirmacher P, Trauner M, Flott-Rahmel B (2011) Histopathological diagnosis of non-alcoholic and alcoholic fatty liver disease. Virchows Archiv 458:511–523

    Article  PubMed  CAS  Google Scholar 

  8. van Werven JR, Marsman HA, Nederveen AJ, Smits NJ, ten Kate FJ, van Gulik TM, Stoker J (2010) Assessment of hepatic steatosis in patients undergoing liver resection: comparison of US, CT, T1-weighted dual-echo MR imaging, and point-resolved 1H MR spectroscopy. Radiology 256:159–168

    Article  PubMed  Google Scholar 

  9. El-Badry AM, Breitenstein S, Jochum W, Washington K, Paradis V, Rubbia-Brandt L, Puhan MA, Slankamenac K, Graf R, Clavien PA (2009) Assessment of hepatic steatosis by expert pathologists: the end of a gold standard. Ann Surg 250:691–697

    Article  PubMed  Google Scholar 

  10. Adams LA, Angulo P (2007) Role of liver biopsy and serum markers of liver fibrosis in non-alcoholic fatty liver disease. Clin Liver Dis 11:25–35, viii.

    Google Scholar 

  11. Contos MJ, Sanyal AJ (2002) The clinicopathologic spectrum and management of nonalcoholic fatty liver disease. Adv Anat Pathol 9:37–51

    Article  PubMed  Google Scholar 

  12. Franzen LE, Ekstedt M, Kechagias S, Bodin L (2005) Semiquantitative evaluation overestimates the degree of steatosis in liver biopsies: a comparison to stereological point counting. Mod Pathol 18:912–916

    Article  PubMed  Google Scholar 

  13. Liquori GE, Calamita G, Cascella D, Mastrodonato M, Portincasa P, Ferri D (2009) An innovative methodology for the automated morphometric and quantitative estimation of liver steatosis. Histol Histopathol 24:49–60

    PubMed  CAS  Google Scholar 

  14. Reeves PG, Nielsen FH, Fahey GC Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123:1939–1951

    PubMed  CAS  Google Scholar 

  15. Fraulob JC, Ogg-Diamantino R, Fernandes-Santos C, Aguila MB, Mandarim-de-Lacerda CA (2010) A mouse model of metabolic syndrome: insulin resistance, fatty liver and non-alcoholic fatty pancreas disease (NAFPD) in C57BL/6 mice fed a high fat diet. J Clin Biochem Nutr 46:212–223

    Article  PubMed  CAS  Google Scholar 

  16. Souza-Mello V, Gregorio BM, Cardoso-de-Lemos FS, de Carvalho L, Aguila MB, Mandarim-de-Lacerda CA (2010) Comparative effects of telmisartan, sitagliptin and metformin alone or in combination on obesity, insulin resistance, and liver and pancreas remodelling in C57BL/6 mice fed on a very high-fat diet. Clin Sci (Lond) 119:239–250

    Article  CAS  Google Scholar 

  17. Vieira VJ, Valentine RJ, Wilund KR, Woods JA (2009) Effects of diet and exercise on metabolic disturbances in high-fat diet-fed mice. Cytokine 46:339–345

    Article  PubMed  CAS  Google Scholar 

  18. Aguila MB, Pinheiro AR, Parente LB, Mandarim-de-Lacerda CA (2003) Dietary effect of different high-fat diet on rat liver stereology. Liver Int 23:363–370

    Article  PubMed  CAS  Google Scholar 

  19. Mandarim-de-Lacerda CA, Fernandes-Santos C, Aguila MB (2010) Image analysis and quantitative morphology. Methods Mol Biol 611:211–225

    Article  PubMed  Google Scholar 

  20. Marques CM, Motta VF, Torres TS, Aguila MB, Mandarim-de-Lacerda CA (2010) Beneficial effects of exercise training (treadmill) on insulin resistance and nonalcoholic fatty liver disease in high-fat fed C57BL/6 mice. Braz J Med Biol Res 43:467–475

    Article  PubMed  CAS  Google Scholar 

  21. Nascimento FA, Barbosa-da-Silva S, Fernandes-Santos C, Mandarim-de-Lacerda CA, Aguila MB (2010) Adipose tissue, liver and pancreas structural alterations in C57BL/6 mice fed high-fat-high-sucrose diet supplemented with fish oil (n − 3 fatty acid rich oil). Exp Toxicol Pathol 62:17–25

    Article  PubMed  CAS  Google Scholar 

  22. Brunt EM (2010) Pathology of nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol 7:195–203

    Article  PubMed  Google Scholar 

  23. Ahishali E, Demir K, Ahishali B, Akyuz F, Pinarbasi B, Poturoglu S, Ibrisim D, Gulluoglu M, Ozdil S, Besisik F, Kaymakoglu S, Boztas G, Cakaloglu Y, Mungan Z, Canberk Y, Okten A (2010) Electron microscopic findings in non-alcoholic fatty liver disease: is there a difference between hepatosteatosis and steatohepatitis? J Gastroenterol Hepatol 25:619–626

    Article  PubMed  CAS  Google Scholar 

  24. Straub BK, Schirmacher P (2010) Pathology and biopsy assessment of non-alcoholic fatty liver disease. Dig Dis 28:197–202

    Article  PubMed  Google Scholar 

  25. Garcia Urena MA, Colina Ruiz-Delgado F, Moreno Gonzalez E, Jimenez Romero C, Garcia Garcia I, Loinzaz Segurola C, Gonzalez P, Gomez Sanz R (1998) Hepatic steatosis in liver transplant donors: common feature of donor population? World J Surg 22:837–844

    Article  PubMed  CAS  Google Scholar 

  26. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR (1999) Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 94:2467–2474

    Article  PubMed  CAS  Google Scholar 

  27. Turlin B, Mendler MH, Moirand R, Guyader D, Guillygomarc’h A, Deugnier Y (2001) Histologic features of the liver in insulin resistance-associated iron overload. A study of 139 patients. Am J Clin Pathol 116:263–270

    Article  PubMed  CAS  Google Scholar 

  28. Sanyal AJ (2002) AGA technical review on nonalcoholic fatty liver disease. Gastroenterology 123:1705–1725

    Article  PubMed  Google Scholar 

  29. Turlin B, Ramm GA, Purdie DM, Laine F, Perrin M, Deugnier Y, Macdonald GA (2009) Assessment of hepatic steatosis: comparison of quantitative and semiquantitative methods in 108 liver biopsies. Liver Int 29:530–535

    Article  PubMed  Google Scholar 

  30. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M, McCullough AJ, Sanyal AJ (2005) Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41:1313–1321

    Article  PubMed  Google Scholar 

  31. Wieckowska A, Feldstein AE (2008) Diagnosis of nonalcoholic fatty liver disease: invasive versus noninvasive. Semin Liver Dis 28:386–395

    Article  PubMed  CAS  Google Scholar 

  32. Lee JH, Kim D, Kim HJ, Lee CH, Yang JI, Kim W, Kim YJ, Yoon JH, Cho SH, Sung MW, Lee HS (2010) Hepatic steatosis index: a simple screening tool reflecting nonalcoholic fatty liver disease. Dig Liver Dis 42:503–508

    Article  PubMed  CAS  Google Scholar 

  33. Lee SS, Park SH, Kim HJ, Kim SY, Kim MY, Kim DY, Suh DJ, Kim KM, Bae MH, Lee JY, Lee SG, Yu ES (2010) Non-invasive assessment of hepatic steatosis: prospective comparison of the accuracy of imaging examinations. J Hepatol 52:579–585

    Article  PubMed  CAS  Google Scholar 

  34. Cruz-Orive LM, Weibel ER (1990) Recent stereological methods for cell biology: a brief survey. Am J Physiol 258:L148–156

    PubMed  CAS  Google Scholar 

  35. Weibel ER (1989) Measuring through the microscope: development and evolution of stereological methods. J Microsc 155:393–403

    Article  PubMed  CAS  Google Scholar 

  36. Souza-Mello V, Mandarim-de-Lacerda CA, Aguila MB (2007) Hepatic structural alteration in adult programmed offspring (severe maternal protein restriction) is aggravated by post-weaning high-fat diet. Br J Nutr 98:1159–1169

    Article  PubMed  CAS  Google Scholar 

  37. Rawlins SR, El-Zammar O, Zinkievich JM, Newman N, Levine RA (2010) Digital quantification is more precise than traditional semiquantitation of hepatic steatosis: correlation with fibrosis in 220 treatment-naive patients with chronic hepatitis C. Dig Dis Sci 55:2049–2057

    Article  PubMed  Google Scholar 

  38. Nakano S, Nagasawa T, Ijiro T, Inada Y, Tamura T, Maruyama K, Kuroda J, Yamazaki Y, Kusama H, Shibata N (2008) Bezafibrate prevents hepatic stellate cell activation and fibrogenesis in a murine steatohepatitis model, and suppresses fibrogenic response induced by transforming growth factor-beta1 in a cultured stellate cell line. Hepatol Res 38:1026–1039

    Article  PubMed  CAS  Google Scholar 

  39. Neves RH, de Barros M, Alencar AC, Costa-Silva M, Aguila MB, Mandarim-de-Lacerda CA, Machado-Silva JR, Gomes DC (2007) Long-term feeding a high-fat diet causes histological and parasitological effects on murine schistosomiasis mansoni outcome. Exp Parasitol 115:324–332

    Article  PubMed  CAS  Google Scholar 

  40. DeLeve LD, Wang X, Kanel GC, Atkinson RD, McCuskey RS (2008) Prevention of hepatic fibrosis in a murine model of metabolic syndrome with nonalcoholic steatohepatitis. Am J Pathol 173:993–1001

    Article  PubMed  CAS  Google Scholar 

  41. Neves RH, Alencar AC, Aguila MB, Mandarim-de-Lacerda CA, Machado-Silva JR, Gomes DC (2007) Light and confocal microscopic observations of adult Schistosoma mansoni from mice fed on a high-fat diet. J Helminthol 81:361–368

    Article  PubMed  Google Scholar 

  42. Tschanz SA, Burri PH, Weibel ER (2011) A simple tool for stereological assessment of digital images: the STEPanizer. J Microsc 243(Pt 1):47–59

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Thatiany Marinho and Angelica Figueiredo for their technical assistance. This research was supported by the Brazilian agencies CNPq (Conselho Nacional de Ciencia e Tecnologia, www.cnpq.br) and FAPERJ (Fundacao para o Amparo a Pesquisa do Estado do Rio de Janeiro, www.faperj.br).

Conflict of interest

The authors declare that they have no conflict interest.

Author information

Authors and Affiliations

  1. Laboratory of Morphometry, Metabolism and Cardiovascular Diseases, Biomedical Center, State University of Rio de Janeiro, Av 28 de Setembro 87 fds, 20551-030, Rio de Janeiro, RJ, Brazil

    Mariana Catta-Preta, Leonardo Souza Mendonca, Julio Fraulob-Aquino, Marcia Barbosa Aguila & Carlos Alberto Mandarim-de-Lacerda

Authors
  1. Mariana Catta-Preta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leonardo Souza Mendonca
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julio Fraulob-Aquino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcia Barbosa Aguila
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carlos Alberto Mandarim-de-Lacerda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carlos Alberto Mandarim-de-Lacerda.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Catta-Preta, M., Mendonca, L.S., Fraulob-Aquino, J. et al. A critical analysis of three quantitative methods of assessment of hepatic steatosis in liver biopsies. Virchows Arch 459, 477–485 (2011). https://doi.org/10.1007/s00428-011-1147-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00428-011-1147-1

Keywords

Search

Navigation