The primary objective of this study was to develop a reliable and reproducible quantification method based on a widely-available cost-free computer software, for the objective evaluation of immunohistochemically-stained tissues. The ImageJ computer-based method developed in this study allows numerical analysis of cells with cell numbers (number of cells/mm2 of tissue) as opposed to previously developed computer-based quantification methods allowing the quantification of the immunostaining intensity .
The intra-operator ICC value confirmed excellent repeatability of the ImageJ method while inter-operator ICC value showed good reproducibility , suggesting that reliability is improved if the method is used by a single operator. This is likely due to the subjective nature of the manual subtraction step required to delete structures considered to be stained non-specifically (Fig. 5d). Few histomorphology studies have validated newly-developed computer-based quantification methods with ICCs to compare the reliability of the methods with the evaluation done by a pathologist or a tissue specialist (e.g. surgeon), mostly considered as the gold standard method [8,9,10,11,12]. To the authors’ knowledge, this study is the first to develop and validate a computer-based quantification method, with ICCs, to evaluate equine wound healing. The ICC found between the ImageJ and the pathologist-based methods (0.67; p < 0.05) confirmed good reliability between the ImageJ and the gold standard methods . Results from this study show no difference in the coefficients of variation between the two methods. The greater mean of coefficients of variation observed in the ImageJ method group could be explained by a single specimen having a coefficient of variation of 96% between count 1 and 2, thus significantly increasing the mean of the group. Other data had a coefficient of correlation between 0 and 40% and had a minimal and maximal difference of 3 and 30%, respectively, with the pathologist group. The manual subtraction step can bring subjectivity and therefore variation between one count to another for the ImageJ method, but the excellent intra-operator ICC found shows that measurements are repeatable. It is difficult to compare such results with other studies aiming to develop computer-based methods applied to cutaneous wound healing since these studies are scarce and ICC agreement intervals may differ in the literature [7, 9, 13, 14]. In short, the hypothesis is partially confirmed since an excellent repeatability and good reproducibility and reliability were found. In light of these results, the newly-developed computer-based ImageJ method presents advantages over manual counting. It yields a precise number of cells in a defined area of tissue and a cell can not be counted twice, limiting human error. Importantly, it requires less expertise and can be used by a variety of personnel when a pathologist or a wound specialist is not available, and it has fewer subjective steps in its process. The ImageJ computer-based method has the manual subtraction step that can introduces subjectivity, while this step as well as the inclusion or exclusion of mast cells contribute to subjectivity in the manual-based counting method. The inclusion or exclusion of mast cells in the counting for the ImageJ method is based on a mathematical parameter (cell area) that is less subjective than morphological parameters like cell shape, visual cell size and nucleus/cytoplasm ratio. It is recognized that in tissues with small numbers of immunostained cells, manual counting could be satisfactory. The advantages discussed above could also make this method a suitable tool for the quantification of other immunostained inflammatory cells like neutrophils and macrophages during physiological and pathological processes. With specific antibodies, the software program can analyze tissues with either numerous or few stained cells since it recognizes the IHC staining. Other mathematical parameters can also be added to the method like the circularity parameter to allow the quantification of multilobed nuclei of cells such as neutrophils [15, 16]. The ImageJ computer-based method we described could eventually help toward monitoring wound healing and response to therapy in the future by the measurement of the immunostained cells present in a small wound biopsy to enhance care.
As a proof of concept, the newly-developed ImageJ method was used to quantitate mast cells, for the first time, in specimens harvested from healing skin wounds of horses. The location of mast cells in the subepidermal and deep dermal layers agrees with other studies conducted on equine skin (Fig. 3A-b) [17, 18]. However, the scattered distribution within the granulation tissue of equine wounds has, to our knowledge, never been reported (Fig. 3A-c). The smaller numbers observed in limb compared to body wounds (Fig. 3b) may reflect a weak inflammatory response seen in limb wounds of horses, refuting our hypothesis . Additional studies are needed to characterize the intrinsic properties of equine mast cells by cell culture and/or other laboratory techniques such as flow cytometry and ELISA to better understand their role in cutaneous wound healing. Although the role of mast cells have been investigated in porcine models where a hypercontractive model was treated with ketotifen (a mast cell stabilizer) and showed significantly less wound contraction than its nontreated control, much remains unknown [19, 20]. Eventually, it may be interesting to compare the kinetics and the number of mast cells of naturally healing equine wounds with those affected by EGT in an effort to establish reference values, a cut off value of mast cell number indicating poor wound prognostic and the clinical role of this cell type.
The principal limitation of our study is the small number of horses, which may explain why no significant differences were found between limb and body wounds in the proof of concept portion of the study, since mast cell numbers are known to show great interindividual variation in horses . However, the multiple sites and the longitudinal nature of the study added robustness for statistical analysis. Results obtained here should help to elucidate the role of mast cells in normal wound healing and future studies might include EGT-affected wounds to document the numbers of mast cells in this healing impairment common to horses.