Feulgen staining applied to nuclear histomorphometry with ImageJ in canine mammary carcinoma subtypes

Authors

Abstract

Histomorphometry is an important tool in the diagnosis and prognosis of breast cancer, enabling the quantification of microscopic features related to tumor aggressiveness. This study aimed to standardize the Feulgen staining method for nuclear morphometric analysis and to compare nuclear measurements among different subtypes of canine mammary carcinoma. Histological slides with tissue microarray (TMA) sections from tubulopapillary carcinoma (TPC), complex carcinoma (CC), and solid carcinoma (SC) diagnoses were stained using the Feulgen method. Five random fields per TMA sample were photographed at 400× magnification, and nuclear morphometry was performed using ImageJ software with the Threshold tool. The SC subtype showed significantly larger nuclear area (85.40 µm² ± 0.3159) and nuclear perimeter (48.33 µm ± 0.1217) compared to the TPC and CC subtypes (P < 0.0001). These results indicate that SC, a high-grade malignant neoplasm, presents greater nuclear dimensions. Nuclear area and perimeter proved to be more reliable parameters for comparisons between different subtypes, while circularity was useful in differentiating malignancy grades within the same subtype. Feulgen staining demonstrated high efficiency for nuclear histomorphometry, allowing precise nuclear delimitation and reducing subjectivity in quantitative evaluation.
Keywords: dog; histochemical staining; malignancy grade; neoplasm; nuclear morphometry.

Keywords: Carcinoma, dog, neoplasms, nuclear morphology.

Downloads

Download data is not yet available.

References

Salas Y, Marquez A, Diaz D, Romero L. Epidemiological Study of Mammary Tumors in Female Dogs Diagnosed during the Period 2002-2012: A Growing Animal Health Problem. PLoS One. 2015;10(5):e0127381. https://doi.org/10.1371/journal.pone.0127381

Zink D, Fischer AH, Nickerson JA. Nuclear structure in cancer cells. Nat Rev Cancer. Sep 2004;4(9):677-87. https://doi.org/10.1038/nrc1430

Goldschmidt M, Pena L, Rasotto R, Zappulli V. Classification and grading of canine mammary tumors. Vet Pathol. Jan 2011;48(1):117-31. https://doi.org/10.1177/0300985810393258

Fischer A H, Jacobson K A, Rose J, Zeller R. Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc. 2008 May 1;2008:pdb.prot4986. https://doi.org/10.1101/pdb.prot4986

Chan JK. The wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology. Int J Surg Pathol. Feb 2014;22(1):12-32. doi: https://doi.org/10.1177/1066896913517939

Bignold LP, Coghlan BL, Jersmann HP. Cancer morphology, carcinogenesis and genetic instability: a background. Exs. 2006;(96):1-24. doi: https://doi.org/10.1007/3-7643-7378-4_1

De Vico G, Maiolino P, Cataldi M, Mazzullo G, Restucci B. Nuclear morphometry in relation to lymph node status in canine mammary carcinomas. Vet Res Commun. Nov 2007;31(8):1005-11. doi: https://doi.org/10.1007/s11259-006-0108-7

Mello MLS, Vidal BC. The Feulgen reaction: A brief review and new perspectives. Acta Histochem. Jul 2017;119(6):603-609. https://doi.org/doi:10.1016/j.acthis.2017.07.002

Lakadamyali M, Lakadamyali M. From feulgen to modern methods: marking a century of DNA imaging advances. Histochemistry and Cell Biology 2024 162:1. 2024-05-16;162(1). doi: https://doi.org/10.1007/s00418-024-02291-z

Elston CW, Ellis IO. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. Nov 1991;19(5):403-10. doi: https://doi.org/10.1111/j.1365-2559.1991.tb00229.x

Cassali GD, Lavalle GE, Ferreira E, et al. Consensus for the diagnosis, prognosis and treatment of canine mammary tumors-2013. 2014. https://repositorio.ufba.br/handle/ri/5386

Pires ARC, Andreiuolo FdM, Souza SRd. TMA for all: a new method for the construction of tissue microarrays without recipient paraffin block using custom-built needles. Diagnostic Pathology. 2006 Jul 25;1(1). doi: https://doi.org/10.1186/1746-1596-1-14

Carella F, De Vico G, Landini G. Nuclear morphometry and ploidy of normal and neoplastic haemocytes in mussels. PLoS One. 2017;12(3)doi: https://doi.org/10.1371/journal.pone.0173219

Papakonstantinou S, O'Brien PJ. "High Content Imaging for the Morphometric Diagnosis and Immunophenotypic Prognosis of Canine Lymphomas". Cytometry B Clin Cytom. Feb 28 2014;doi: https://doi.org/10.1002/cytob.21170

Simeonov R, Simeonova G. Computerized cytomorphometric analysis of nuclear area, nuclear perimeter and mean nuclear diameter in spontaneous canine mammary gland tumours. Vet Res Commun. Jul 2007;31(5):553-8. doi: https://doi.org/10.1007/s11259-007-3562-y

Destexhe E, Bicker E, Coignoul F. Image analysis evaluation of ploidy, S-phase fraction and nuclear area in canine mammary tumours. Journal of Comparative Pathology. 1995/10/01;113(3)doi: https://doi.org/10.1016/S0021-9975(05)80036-2

Cassali GD, Bertagnolli AC, Gärtner F, Schmitt F. Canine mammary tumours: A quantitative DNA study using static cytometry. Revista Española de Patología. 2011/10/01;44(4)doi: https://doi.org/10.1016/j.patol.2011.05.005

El Din AA, Badawi MA, Aal SE, Ibrahim NA, Morsy FA, Shaffie NM. DNA Cytometry and Nuclear Morphometry in Ovarian Benign, Borderline and Malignant Tumors. Open Access Maced J Med Sci. Dec 15 2015;3(4):537-44. doi: https://doi.org/10.3889/oamjms.2015.104

Yang X, Xiao X, Wu W, et al. Cytological study of DNA content and nuclear morphometric analysis for aid in the diagnosis of high-grade dysplasia within oral leukoplakia. Oral Surg Oral Med Oral Pathol Oral Radiol. Sep 2017;124(3):280-285. doi: https://doi.org/10.1016/j.oooo.2017.05.509

Kesarkar K, Tamgadge A, Peirera T, Tamgadge S, Gotmare S, Kamat P. Evaluation of Mitotic Figures and Cellular and Nuclear Morphometry of Various Histopathological Grades of Oral Squamous Cell Carcinoma: Comparative study using crystal violet and Feulgen stains. Sultan Qaboos Univ Med J. May 2018;18(2):e149-e154. doi: https://doi.org/10.18295/squmj.2018.18.02.005

De Potter CR, Praet MM, Slavin RE, Verbeeck P, Roels HJ. Feulgen DNA content and mitotic activity in proliferative breast disease. A comparison with ductal carcinoma in situ. Histopathology. Dec 1987;11(12):1307-19.

Diamond DA, Berry SJ, Jewett HJ, Eggleston JC, Coffey DS. A new method to assess metastatic potential of human prostate cancer: relative nuclear roundness. J Urol. Oct 1982;128(4):729-34. doi: https://doi.org/10.1016/s0022-5347(17)53158-4

Andrea Carlos Eduardo, Bleggi-Torres Luiz Fernando, Seixas AMTd. Análise da morfometria nuclear: descrição da metodologia e o papel dos softwares de edição de imagem. J Bras Patol Med Lab. 2008;44: 51-57. doi: http://dx.doi.org/10.1590/S1676-24442008000100010.

Rueden CT, Schindelin J, Hiner MC, et al. ImageJ2: ImageJ for the next generation of scientific image data. OriginalPaper. BMC Bioinformatics. 2017-11-29 2017;18(1):1-26:doi: https://doi.org/10.1186/s12859-017-1934-z

Burrai GP, Gabrieli A, Moccia V, et al. A Statistical Analysis of Risk Factors and Biological Behavior in Canine Mammary Tumors: A Multicenter Study. Animals : an Open Access Journal from MDPI. 2020 Sep 18;10(9)doi: https://doi.org/10.3390/ani10091687

Silva EMGd, Santos TRd, Silva MJB, da Silva EMG, dos Santos TR, Silva MJB. Identifying the Risk Factors for Malignant Mammary Tumors in Dogs: A Retrospective Study. Veterinary Sciences 2023, Vol 10, Page 607. 2023-10-05;10(10)doi: https://doi.org/10.3390/vetsci10100607

Rodríguez J, Santana Á, Herráez P, Killick DR, Monteros AEdl. Epidemiology of canine mammary tumours on the Canary Archipelago in Spain. BMC Veterinary Research. 2022 Jul 11;18(1)doi: https://doi.org/10.1186/s12917-022-03363-9

Howell A, Bleehen NM, Taylor PJ. Nuclear morphometry and glutathione S-transferase pi expression in breast cancer. Oncol Rep. 2000 May-Jun;7(3):525–528. https://doi.org/10.3892/or.7.3.609

Rasotto R, Berlato D, Goldschmidt MH, Zappulli V. Prognostic Significance of Canine Mammary Tumor Histologic Subtypes: An Observational Cohort Study of 229 Cases. Vet Pathol. Jul 2017;54(4):571-578. doi: https://doi.org/10.1177/0300985817698208

Kashyap A, Jain M, Shukla S, Andley M. Study of nuclear morphometry on cytology specimens of benign and malignant breast lesions: A study of 122 cases. J Cytol. Jan-Mar 2017;34(1):10-15. doi: https://doi.org/10.4103/0970-9371.197591

Di Donato G, Laufer-Amorim R, Palmieri C. Nuclear morphometry in histological specimens of canine prostate cancer: Correlation with histological subtypes, Gleason score, methods of collection and survival time. Res Vet Sci. Oct 2017;114:212-217. doi: https://doi.org/10.1016/j.rvsc.2017.05.007

Nativ O, Sabo E, Reiss R, Moskovitz B, Melamed M, Gutman M. The role of nuclear morphometry for predicting disease outcome in patients with localized renal cell carcinoma. Cancer. 1995 Oct 15;76(8):1447–1452. doi: https://doi.org/10.1002/1097-0142

Andrea CE, Petrilli AS, Jesus-Garcia R, Bleggi-Torres LF, MT. A. Large and Round Tumor Nuclei in Osteosarcoma: Good Clinical Outcome. International journal of clinical and experimental pathology. 01/30/2011 2011;4(2)

Biggiogera M, Cavallo M, Casali C. A brief history of the Feulgen reaction. Histochemistry and Cell Biology. 2024 Apr 12;162(1-2)doi: https://doi.org/10.1007/s00418-024-02279-9

Published

2025-10-28

How to Cite

RODRIGUES, Arthur Perillo; CARVALHO, Mara Taís de; FERREIRA, Hugo Henrique; MENEZES, Liliana Borges de; MIGUEL, Marina Pacheco. Feulgen staining applied to nuclear histomorphometry with ImageJ in canine mammary carcinoma subtypes. Brazilian Animal Science/ Ciência Animal Brasileira, Goiânia, v. 26, 2025. Disponível em: https://revistas.ufg.br/vet/article/view/82640. Acesso em: 5 dec. 2025.

Issue

Section

VETERINARY MEDICINE

Data statement

  • The research data is available on demand, condition justified in the manuscript