Development of a computer-based quantification method for immunohistochemically-stained tissues and its application to study mast cells in equine wound healing (proof of concept).
Abstract: There is a growing interest in the scientific community to use computer-based software programs for the quantification of cells during physiological and pathophysiological processes. Drawbacks of computer-based methods currently used to quantify immunohistochemical staining are the complexity of use, expense of software and overly-simplified descriptions of protocol thereby limiting reproducibility. The precise role of mast cells in equine cutaneous wound healing is unknown. Given the contribution of mast cells to the chronic inflammation observed in human keloid, a pathology similar to exuberant granulation tissue (EGT) in horses, mast cells might be present in high numbers in equine limb wounds predisposed to EGT. The main goal of this study was to develop a reliable and reproducible quantification method for immunostained tissues using a computer software that is widely available, at no cost, to the scientific community. A secondary goal was to conduct a proof of concept using the newly-established method to quantify mast cells during wound healing at different anatomical sites (body and limb) in horses to see if a different pattern is observed in limb wounds, which are predisposed to EGT. Results: A good intraclass correlation coefficient (ICC, 0.67 p < 0.05) was found between the computer-based ImageJ method and manual counting. An excellent intra-operator ICC of 0.90 (p < 0.01) was found for the ImageJ quantification method while a good interoperator ICC of 0.69 (p < 0.01) was measured. No significant difference was observed between the variation of the ImageJ and that of the manual counting method. Mast cells were localized below the epidermis, around cutaneous appendages and blood vessels. Mast cell numbers did not differ significantly in relation to anatomical location or time of healing. Conclusions: The computer-based quantification method developed is reliable, reproducible, available, cost-free and could be used to study different physiological and pathological processes using immunohistochemistry.
Publication Date: 2020-07-02 PubMed ID: 32616050PubMed Central: PMC7330934DOI: 10.1186/s12917-020-02444-xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research created a reliable method using free computer software to quantify cells in tissue samples stained for imaging (immunohistochemistry). The researchers also tested this method on samples from horses recovering from wounds, specifically tracking mast cells—a type of immune cell—across different wound locations and stages of healing.
Objective of the Study
- The primary objective of the study was to create a computer-based method for quantifying specific cells in immunohistochemically stained tissue samples. The researchers aimed to overcome issues with existing methods, such as high cost, complexity of use, and low reproducibility due to simplistic protocol descriptions.
- The secondary objective was to apply this new method to investigate the role of mast cells in equine wound healing, particularly in wounds predisposed to Exuberant Granulation Tissue (EGT), a condition similar to human keloid scars.
Methodology and Testing
- The software used in the development of this method was ImageJ, a cost-free image processing program often used in scientific research.
- Reliability of the method was tested by comparing the results of the computer-based cell quantification with manual counting. The consistency (intra-operator Intraclass Correlation Coefficient, ICC) of using the ImageJ method, and the variation between different operators (inter-operator ICC) were evaluated.
- Focusing on equine wound healing, the researchers used this new method to quantify mast cells in different anatomical sites and at different stages of wound healing.
Results
- The ImageJ method showed a substantial agreement with manual counting (ICC 0.67), and excellent consistency between repeated measurements by the same person (intra-operator ICC of 0.90), indicating the method’s reliability.
- The ImageJ method’s variation between different operators also showed a good degree of agreement (inter-operator ICC of 0.69).
- In the study of equine wound healing, mast cells were found localized below the skin’s surface, around skin appendages, and blood vessels. However, the number of mast cells did not vary significantly depending on the location of the wound or the time of healing.
Conclusion
- The computer-based cell quantification method developed in this study proved to be reliable, reproducible, freely available, and cost-free. It could be a useful tool in future studies of physiological and pathological processes using immunohistochemistry.
- Although the role of mast cells in equine wound healing is still unclear, the method provided valuable insights into their localization during this process.
Cite This Article
APA
Dubuc V, Laverty S, Richard H, Doré M, Theoret C.
(2020).
Development of a computer-based quantification method for immunohistochemically-stained tissues and its application to study mast cells in equine wound healing (proof of concept).
BMC Vet Res, 16(1), 228.
https://doi.org/10.1186/s12917-020-02444-x Publication
Researcher Affiliations
- Department of Veterinary Biomedicine, Faculté de Médecine Vétérinaire, Université de Montréal, 3 200 Sicotte, Saint-Hyacinthe, Québec, Canada. valerie.dubuc@umontreal.ca.
- Department of Clinical Sciences, Faculté de Médecine Vétérinaire, Université de Montréal, 1 500 des Vétérinaires, Saint-Hyacinthe, Québec, Canada.
- Department of Clinical Sciences, Faculté de Médecine Vétérinaire, Université de Montréal, 1 500 des Vétérinaires, Saint-Hyacinthe, Québec, Canada.
- Department of Pathology and Microbiology, Faculté de Médecine Vétérinaire, Université de Montréal, 3 200 Sicotte, Saint-Hyacinthe, Québec, Canada.
- Department of Veterinary Biomedicine, Faculté de Médecine Vétérinaire, Université de Montréal, 3 200 Sicotte, Saint-Hyacinthe, Québec, Canada.
MeSH Terms
- Animals
- Cell Count / veterinary
- Granulation Tissue / cytology
- Granulation Tissue / diagnostic imaging
- Horses
- Image Processing, Computer-Assisted / standards
- Immunohistochemistry / methods
- Immunohistochemistry / veterinary
- Mast Cells / cytology
- Software / standards
- Wound Healing
Grant Funding
- NSERC 250231 / Natural Sciences and Engineering Research Council of Canada
Conflict of Interest Statement
No competing interests have been declared.
References
This article includes 23 references
- Choudhury KR, Yagle KJ, Swanson PE, Krohn KA, Rajendran JG. A robust automated measure of average antibody staining in immunohistochemistry images.. J Histochem Cytochem 2010;58(2):95–107.
- Jensen EC. Quantitative analysis of histological staining and fluorescence using ImageJ.. Anatomical Rec (Hoboken, NJ : 2007) 2013;296(3):378–381.
- Wilmink JM, Veenman JN, van den Boom R, Rutten VP, Niewold TA, Broekhuisen-Davies JM, Lees R, Armstrong S, van Weeren PR, Barneveld A. Differences in polymorphonucleocyte function and local inflammatory response between horses and ponies.. Equine Vet J 2003;35(6):561–569.
- Shaker SA, Ayuob NN, Hajrah NH. Cell talk: a phenomenon observed in the keloid scar by immunohistochemical study.. Appl Immunohistochem Mol Morphol 2011;19(2):153–159.
- Ng MF. The role of mast cells in wound healing.. Int Wound J 2010;7(1):55–61.
- Matos LL, Stabenow E, Tavares MR, Ferraz AR, Capelozzi VL, Pinhal MA. Immunohistochemistry quantification by a digital computer-assisted method compared to semiquantitative analysis.. Clinics (Sao Paulo, Brazil) 2006;61(5):417–424.
- Cicchetti DV, Sparrow SA. Developing criteria for establishing interrater reliability of specific items: applications to assessment of adaptive behavior.. Am J Ment Defic 1981;86(2):127–137.
- Prieto MF, Acha B, Gomez-Cia T, Fondon I, Serrano C. A system for 3D representation of burns and calculation of burnt skin area.. Burns 2011;37(7):1233–1240.
- Straughen JK, Misra DP, Ernst LM, Charles AK, VanHorn S, Ghosh S, Buhimschi I, Buhimschi C, Divine G, Salafia CM. Methods to decrease variability in histological scoring in placentas from a cohort of preterm infants.. BMJ Open 2017;7(3):e013877.
- Wang SC, Anderson JAE, Evans R, Woo K, Beland B, Sasseville D, Moreau L. Point-of-care wound visioning technology: reproducibility and accuracy of a wound measurement app.. PLoS One 2017;12(8):e0183139.
- Malone M, Schwarzer S, Walsh A, Xuan W, Al Gannass A, Dickson HG, Bowling FL. Monitoring wound progression to healing in diabetic foot ulcers using three-dimensional wound imaging.. J Diabetes Complicat 2020;34(2):107471.
- Koetsier KS, Wong JN, Muffley LA, Carrougher GJ, Pham TN, Gibran NS. Prospective observational study comparing burn surgeons' estimations of wound healing after skin grafting to photo-assisted methods.. Burns 2019;45(7):1562–1570.
- O'Connor SR, Tully MA, Ryan B, Bradley JM, Baxter GD, McDonough SM. Failure of a numerical quality assessment scale to identify potential risk of bias in a systematic review: a comparison study.. BMC Res Notes 2015;8:224.
- Cuchna JW, Hoch MC, Hoch JM. The interrater and intrarater reliability of the functional movement screen: a systematic review with meta-analysis.. Phys Ther Sport 2016;19:57–65.
- Ulbrich H, Prech P, Luxenburger A, Dannhardt G. Characterization of a computerized assay for rapid and easy determination of leukocyte adhesion to endothelial cells.. Biol Pharm Bull 2005;28(4):718–724.
- Garrity-Park M, Loftus EV Jr, Sandborn WJ, Smyrk TC. Myeloperoxidase immunohistochemistry as a measure of disease activity in ulcerative colitis: association with ulcerative colitis-colorectal cancer, tumor necrosis factor polymorphism and RUNX3 methylation.. Inflamm Bowel Dis 2012;18(2):275–283.
- Scott DW. Equine dermatology.. 2. St. Louis: Saunders; 2011.
- Jorgensen E, Lazzarini G, Pirone A, Jacobsen S, Miragliotta V. Normal microscopic anatomy of equine body and limb skin: a morphological and immunohistochemical study.. Ann Anat 2018;218:205–212.
- Kawarai S, Masuda K, Ohmori K, Matsuura S, Yasuda N, Nagata M, Sakaguchi M, Tsujimoto H. Cultivation and characterization of canine skin-derived mast cells.. J Vet Med Sci 2010;72(2):131–140.
- Gallant-Behm CL, Hildebrand KA, Hart DA. The mast cell stabilizer ketotifen prevents development of excessive skin wound contraction and fibrosis in red Duroc pigs.. Wound Repair Regen 2008;16(2):226–233.
- Kamus L, Rameau M, Theoret C. Feasibility of a disposable canister-free negative-pressure wound therapy (NPWT) device for treating open wounds in horses.. BMC Vet Res 2019;15(1):78.
- Bigbie RB, Schumacher J, Swaim SF, Purohit RC, Wright JC. Effects of amnion and live yeast cell derivative on second-intention healing in horses.. Am J Vet Res 1991;52(8):1376–1382.
- Theoret CL, Olutoye OO, Parnell LK, Hicks J. Equine exuberant granulation tissue and human keloids: a comparative histopathologic study.. Vet Surg 2013;42(7):783–789.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
