Computer assisted image analysis in the diagnosis of gynaecological lesions A quantitative and comparative investigation of haematoxylin eosin with special dyes on tissue lable at ScienceDirect Journa[.]
Journal of Cancer Research and Practice xxx (2016) 1e9 Contents lists available at ScienceDirect Journal of Cancer Research and Practice journal homepage: http://www.journals.elsevier.com/journal-of-cancerresearch-and-practice Original Article Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue F.M Onyije a, b, *, A.A Ngokere b, A.E Ligha c, O.O Mgbere d, O.G Avwioro e a Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria b Department of Medical Laboratory Science, Faculty of Health Science and Technology, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria c Department of Human Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria d Institute of Community Health, University of Houston, Texas Medical Center, Houston, TX, USA e Faculty of Science, Delta State University, Abraka, Nigeria a r t i c l e i n f o a b s t r a c t Article history: Received 24 July 2016 Accepted November 2016 Available online xxx Background: Morphometry and quantitative image analysis have shown considerable potential for diagnostic applications because they eliminate subjectivity and increase the measurement reproducibility In our study, we investigated the use of morphometric quantitative image analysis as a tool to aid diagnosis of lesion tissues stained with seven staining techniques Material and methods: Thirteen already established lesions were examined in this study Tissues were sectioned at mm thickness using the rotary microtome and prepared for staining according to standard methods before staining them with seven different histochemical dyes: Haematoxylin and Eosin (H&E), Masson's Trichrome (MT), Periodic Acid Schiff (PAS), Phosphotungstic Acid Haematoxylin (PTAH), Southgate Mucincamine (SGM), Alcian Blue (AB) and Verhoeff Van Gieson (VVG) Photomicrographs were analyzed using imageJ for percentage area and intensity measurement Results: The average % area and intensity measurement based on the seven staining techniques used in this study were 47.88% and 122.23 points (pts) The lesions, adenomyosis in MT (80%), squamous cell carcinoma in PAS (80%) and fibroma in PTAH (77%) stained above the average % area The squamous cell carcinoma stained with MT was the most intense tissue (55 pts), followed by Brenner tumour in PTAH (61 pts) and squamous cell carcinoma in PAS (69 pts) There were significant correlations between the % area of tissues covered by PAS and H&E (r ¼ 0.599, p ¼ 0.030), and PAS and MT (r ¼ 0.572, p ¼ 0.041) as well as in the intensity of tissues in PAS and H&E (r¼0.615, p¼0.025), VVG and H&E (r ¼ 0.707, p ¼ 0.007), and VVG and PTAH (r ¼ 0.577, p ¼ 0.039) Conclusion: MT staining technique produced the best results in the % area of tissues covered and the intensity measurements, and therefore was recommended for routine use alongside with H&E in diagnostic histopathology © 2016 The Chinese Oncology Society Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Keywords: Gynaecological lesion Dye Morphometry Computerized Quantitative Analysis Image analysis Introduction * Corresponding author Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria E-mail address: onyijefelix@yahoo.com (F.M Onyije) Peer review under responsibility of The Chinese Oncology Society Histopathology involves the use of dyes for microscopic examination of tissues in part to spot signs and characteristic features of diseases Recently, computer assisted diagnosis (CAD) has been widely used in developed countries.1e3 In general, it is wellrecognised that the importance of quantitative analysis in histopathology cannot be overemphasized, particularly because most pathology diagnoses are based on the subjective opinion of http://dx.doi.org/10.1016/j.jcrpr.2016.11.002 2311-3006/© 2016 The Chinese Oncology Society Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 pathologists.4 The utilization of CAD has the potential to improve the practice of histopathology and other related discipline in different ways.5 With the influx of different imaging technologies, histological images of diverse staining techniques including immunehistochistry are produced for either manual or automated analysis Generally, such images are better obtained at low magnification.6 ImageJ, which is a public domain, Java-based image processing program developed at the National Institute of Health,7,8 is used in quantitative analysis of histological stains on tissues, among other functions.9 Although the human eye is sensitive to a number of factors, including luminosity and variation in contrast and brightness, critical visual analysis is open to subjective interpretation The essence of morphometry is to eliminate subjectivity and increase the reproducibility of measurements.10 The quantification of percentage area covered by different stains on different tissues as well as the intensity measurement have not been given due attention, a situation that has resulted in the lack of improvement in routine staining procedures Advances in computer image analysis techniques allow more accurate quantification of histopathological analysis.11 The mechanisms of tissue staining are histochemical in nature, which is the application of chemical substances onto a tissue in order to produce a visible outcome To achieve this reaction, the tissue takes up the stain either through a chemical or physical method (adsorption, absorption, solubility, osmotic pressure or capillary attraction) The type and nature of this uptake may differ from one tissue to another, as well as by the technique employed There are two groups of dyes commonly used: 1) the natural group of dyes, which are derived from plants and insects, 2) the synthetic dyes, which are derived from coal tar These groups of dyes can be applied regressively (differentiation), progressively (without differentiation) or directly (without the use of mordant).12 The importance of histopathological staining cannot be overemphasized, especially as the process allows for adequate and reliable histological results to be obtained Hence, in this study, we investigated the use of morphometric quantitative image analysis as a tool to aid in the diagnosis of stained lesions using seven staining techniques Materials and methods 2.1 Tissue Ninety-one (91) cut sections from thirteen (13) different established lesions retrieved from archives of the Histopathology Laboratory at Braithwaite Memorial Specialist Hospital, Port Harcourt, Nigeria were used for this analysis The tissues were sectioned at mm thickness using the rotary microtome and prepared for staining, using the methods described by Suvarna et al.13 Fig Chart showing the % area measurement of lesions within stain methods Haematoxylin and Eosin (H&E), Masson's Trichrome (MT), Periodic Acid Schiff (PAS), Phosphotungstic Acid Haematoxylin (PTAH), Southgate Mucincamine (SGM), Alcian Blue (AB) and Verhoeff Van Gieson (VVG) Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 2.2 Staining methods The staining techniques used in this study were; Haematoxylin and Eosin,14 Masson's Trichrome,15 Periodic Acid Schiff,16 Phosphotungstic Acid Haematoxylin,17 Southgate Mucincamine,18 Alcian Blue19 and Verhoeff Van Gieson20 2.3 Principle of H&E This is the oxidation of haematoxylin to haematein by natural or chemical agent and characteristically requires a mordant for its effectiveness The haematein dye binds to acidic components of the tissue that results in a blue coloration On the other hand, eosin stains pink and has the ability to bind to basic components due to its acidic nature 2.4 Principle of PTAH The phosphotungstic acid binds to the available haematein to form a blue lake pigment, which stains the muscle cross striations, fibrin, nuclei, and other tissue elements blue, Phosphotungstic acid stains collagen fibres red to brown 2.5 Principle of PAS Periodic acid oxidizes 1:2 glycol groups to yield aldehydes, which thereafter recolor Schiff's reagent The principle of the Schiff’’s reaction depends on the conversion or loss of the quinoid structure and the masking of chromophores This forms a colorless compound called leuco-fuchsin which can change to the classic red reaction when exposed to aldehydes Excess Schiff reagent is removed by potassium metabisulfite thus preventing a false positive by oxidation of the reagent in the tissue 2.6 Principle of AB Alcian blue consists of a group of polyvalent basic dyes that are characteristically water-soluble, whose blue color can be attributed to the presence of copper in the molecule It stains sulphated mucoploysaccharides at pH 1, and stains both sulphated and carboxylated mucopolysaccharides at pH 2.5 2.7 Principle of MT As the name implies, three dyes are employed selectively staining muscle, collagen fibers, fibrin, and erythrocytes The general rule in trichrome staining is that less porous tissues are coloured by the smallest dye molecule; thus, whenever a dye of large molecular size penetrates, it will always so at the expense of the smaller molecule Others suggest that the tissue is stained first with Fig Chart showing the intensity measurement of lesions within stain methods Haematoxylin and Eosin (H&E), Masson's Trichrome (MT), Periodic Acid Schiff (PAS), Phosphotungstic Acid Haematoxylin (PTAH), Southgate Mucincamine (SGM), Alcian Blue (AB) and Verhoeff Van Gieson (VVG) Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 the acid dye Biebrich Scarlet, which binds with acidophilic tissue components Thereafter, when treated with phospho amino acids, the less permeable components retain the red stain, while the red stain is extracted from the collagen Simultaneously, this causes a link to which collagen can bind 2.8 Principle of SGM built-in 3.0 MP digital camera compound LED Binocular Microscope The histologic image characteristics of the stains were analyzed and the stain intensity and percentage (%) area stained were measured using ImageJ 1.48 version (National Institute of Health, USA) 2.11 Percentage (%) area and intensity measurement Aluminum is believed to form a chelation complex with carmine, changing the molecule to a positive charge and allowing it to bind with the acid substrates of low density (such as mucins) 2.9 Principle of VVG For VVG, the tissue is stained with a regressive haematoxylin, consisting of ferric chloride and iodine The differentiation is accomplished by using excess mordant (ferric chloride) to break the tissue-mordant dye complex The dye is attracted to the larger amount of mordant in the differentiating solution and is removed from the tissue Elastic tissue has the strongest affinity of the ironhaematoxylin complex, and retains the dye longer than other tissue elements 2.10 Microscopy and data acquisition from photomicrographs The stained tissue slides were viewed using OMAX 40X-2000X Imported RGB images are converted to gray scale images on ImageJ The software quantifies the staining intensity by measuring the pixel value of each pixel in grayscale images following a threshold of areas of staining activity, and converting the pixel value to brightness value or gray value, in a scale of 0e255 from the less bright (that is lower points (pts) and greater intensity) to brighter (that is higher points and reduced intensity) 2.12 Statistical analysis 2.12.1 Analytical package used Measurement system analyses were performed to compare the staining methods by lesions using JMP statistical discovery™ software, version 12.0 (SAS Institute, Cary, NC, USA) For all tests performed, the probability value of 0.05 was used as the threshold for determining statistical significance level Fig Partition flowchart for % area by stain methods and lesions group Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 endometrial polyp in AB (181 pts) were the least intense lesions observed in our study 2.13 Ethical approval Ethical approval for this study was obtained from the Hospital Management Board of Rivers State through the ethics committee of BMSH Results 3.1 Percentage (%) area measurement Fig 1, shows the average % area (48%) of the lesions covered by stains Adenomyosis in MT (80%) followed by squamous cell carcinoma in PAS (80%) and fibroma in PTAH (77%) were above the average percentage area However, the % area of condyloma in AB (17%), adenocarcinoma in PAS (18%) and fibroma in SGM (20%) were generally lower than the average % area in our study 3.2 Intensity measurement Fig 2, shows the mean intensity measurement (122.23 pts) for the different lesions impacted by the different stains Lesions in MT, PTAH and H&E were highly intense, with squamous cell carcinoma in MT (55 pts) and Brenner tumour in PTAH (61 pts) being the most intense lesions Contrarily, mucinous cyst in PAS (201 pts) and 3.3 Partition flowchart Fig 3, shows the partition flow chart of the staining technique and the lesion group The lesion group stained with VVG, PATH and MT had a highest mean % area of 57%, while those stained with SGM, PAS and H&E recorded a lower mean % area of 41% The group with the higher average % area recorded 61% when stained in the right split (fibroma, condyloma, adenomyosis, squamous cell carcinoma, mucinous cyst, leiomyoma, Brenner tumour and adenoma) The left split consisting of demoid cyst, choriocarcinoma, adenocarcinoma and endometrial hyperplasia of the same group recorded 50% Based on the SGM, PAS and H&E staining techniques, the following lesions; endometrial polyp, squamous cell carcinoma, leiomyoma, Brenner tumour and adenoma recorded an average % area of 49% on the right split Thirty two sections of the tissue samples in the left split of the flowchart recorded the mean % area of 35% Fig 4, shows the partition flowchart for intensity measurement by stain methods and lesions group Based on our analysis two main groups were identified as follows; MT and PTAH (group 1), which recorded overall average intensity measurement of 84 pts compared to H&E, VVG, SGM, PAS and AB (group 2), which produced an average intensity Fig Partition flowchart for intensity measurement by stain methods and lesions Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 Fig Matched pairs analysis of percentage area of stains measurement of 137 pts However, subsequent assessment indicates that squamous cell carcinoma, Brenner tumour, mucinous cyst, condyloma, fibroma, endometrial polyp and choriocarcinoma when stained using techniques in group produced a more intense measurement (74 pts) compared to 96 pts obtained when demoid cyst, adenoma leiomyoma, endometrial hyperplasia, adenomyosis and adenocarcinoma were stained using methods in group 3.4 Matched pairs analysis of percentage area of stains Fig 5, presents the matched pairs analysis of % area measurements by staining methods Using H&E as the standard (mean ¼ 42.6%), we compared the six staining methods Results from this analysis indicate that the mean % areas of tissues covered were significantly improved by 50% with MT (64.4%, p ¼ 0.0002), 27% with PTAH (54.2%, p ¼ 0.021) and 26% with VVG (53.7%, p ¼ 0.004) when compared to H&E However, SGM, PAS and AB performed poorly by producing mean % areas that were below or failed to match that of H&E (p > 0.005) 3.5 Matched pairs analysis of stains intensity Fig 6, shows the matched pairs analysis of the intensity measurements by staining methods Tissues stained using MT and PTAH techniques produced the most intense mean measurements that were 34% (80.9 pts, p ¼ 0.0001) and 29% (87.3 pts, p ¼ 0.0001) better than those produced from tissues stained with H&E, which served as the standard On the other hand, intensity measurement for tissues stained with AB and PAS performed poorly being 33% (162.9 pts, p ¼ 0.0001) and 18% (144.7 pts, p ¼ 0.009) less intense than those stained with H&E 3.6 Pair-wise correlation of staining methods Tables and 2, show the pair-wise correlation of % area and intensity measurement by staining methods With reference to % area, except for significant correlations noted between PAS and H&E (r ¼ 0.599, p ¼ 0.030) and PAS and MT (r ¼ 0.572, p ¼ 0.041), all other staining methods were not statistically significant (p > 0.05) Similarly, our study noted significant correlations in intensity measurements by staining methods as follows; PAS and H&E (r ¼ 0.615, p ¼ 0.025), VVG and H&E (r ¼ 0.707, p ¼ 0.007) and, VVG and PTAH (r ¼ 0.577, p ¼ 0.038) Discussion The % area of the tissue covered by stain as well as the intensity is dependent on the type of stain and tissue Stain uptake is often due to dye-tissue or reagent-tissue affinities, permeability and pore size MT covered a greater % area with a high intensity measurement for most tissues compared to other type of staining methods used in our study This was reflected in the outcome of the flow chart (Fig 3), where it was observed that VVG, PTAH and MT occupied a larger surface area than SGM, PAS Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 Fig Matched pairs analysis of intensity of stains and H&E MT was originally used for the demonstration of connective and mesenchymal tissues,13 with the ability to impact up to three colours to tissue and bind with collagen fibres.24 Suvarna et al.13 stated that smaller molecule dyes will stain any of the basic type of tissues This is similar to the rule of trichrome staining, which states that a smaller dye molecule will penetrate and stain tissue element, but whenever a larger dye molecule can penetrate the same element, the smaller molecule will be displaced by it.21 Other factors that contribute to dye-tissue affinity include Coulombic attraction, which is also referred to as salt links or electrostatic bonds Also, Van der Waals forces including intermolecular attractions, hydrogen bonding, covalent bonding, and the hydrophobic effect are among other contributing factors.22,23,13 In our study we observed that MT produced a 50% improvement in the % area of tissues covered over that of H&E It is possible that its three-in-one (trichrome) combination of dyes may be responsible for the large % area covered and high intensity measurement This is in line with the report of Street et al.,25 who stated that MT is a widely used stain in Histology MT is primarily used to enhance the visibility of collagen and elastic fibres and most tissues in the body contain these fibres Adenomyosis refers to the growth of the basal layer of the endometrium into the myometrium.26 The elevated % area covered by MT in adenomyosis is an indication that adenomyosis muscle cells have the capacity to absorb stain and has a large surface area, as a result of its dense fascicles with elongated cytoplasm and nuclei having a finely dispersed chromatin.27 Squamous cell carcinoma was the most covered and intensified tissue in our study, which may be due to the numerous malignant cells and their metachromatic nature Our observation is in line with the publication of the British Association for the Advancement of Science where it was noted that malignant cells take up stains two to three times more than the normal cells.28 This affirms why malignant lesions had a higher intensity and % area measurement than the benign lesions with MT These findings support the work of Ouyang et al.,29 who reported that MT is superior to H&E in the examination of tissue sections, and concluded that MT would be a valuable tool for analysing autopsy tissues The % area covered by stains and intensity measurement aid in the visualization of cells and tissues However, poor coverage and intensity may obscure accurate and reliable diagnosis Conclusion The quantitative and comparative analysis of MT in both % area and intensity measurement with other techniques in this study clearly demonstrates that MT is better than other staining methods Therefore, MT was recommended for routine use alongside with H&E in diagnostic histopathology Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 Table Pair-wise correlation of % area measurements by stain methods Variable By variable Correlation Count Lower 95% Upper 95% Significant probability H&E AB 0.1110 13 0.4686 0.6239 0.7180 MT AB 0.1138 13 0.4664 0.6256 0.7113 MT H&E 0.1069 13 0.4719 0.6213 0.7281 PAS AB 0.0956 13 0.6143 0.4807 0.7559 PAS H&E 0.5993 13 0.0722 0.8648 0.0304* PAS MT 0.5722 13 0.0310 0.8540 0.0410* PTAH AB 0.1973 13 0.6749 0.3968 0.5183 PTAH H&E 0.0054 13 0.5472 0.5548 0.9859 PTAH MT 0.0343 13 0.5744 0.5266 0.9114 PTAH PAS 0.0192 13 0.5642 0.5375 0.9503 SGM AB 0.2247 13 0.3724 0.6902 0.4605 SGM H&E 0.1869 13 0.4059 0.6690 0.5410 SGM MT 0.1273 13 0.4557 0.6338 0.6786 SGM PAS 0.1945 13 0.3993 0.6733 0.5242 SGM PTAH 0.0611 13 0.5921 0.5070 0.8428 VVG AB 0.2153 13 0.3809 0.6850 0.4800 VVG H&E 0.5106 13 0.0562 0.8285 0.0746 VVG MT 0.1407 13 0.4448 0.6419 0.6467 VVG PAS 0.3383 13 0.2614 0.7496 0.2582 VVG PTAH 0.1608 13 0.6538 0.4282 0.5998 VVG SGM 0.4587 13 0.1235 0.8060 0.1149 Graphical representation Table Pair-wise correlation of intensity measurements by stain methods Variable by Variable Correlation Count Lower 95% Upper 95% Significant Probability H&E AB 0.0733 13 0.4978 0.6000 0.8120 MT AB 0.1744 13 0.6618 0.4166 0.5688 MT H&E 0.2200 13 0.3766 0.6876 0.4702 PAS AB 0.3723 13 0.7661 0.2248 0.2103 PAS H&E 0.6147 13 0.0964 0.8708 0.0254* PAS MT 0.2783 13 0.3221 0.7190 0.3572 PTAH AB 0.0126 13 0.5597 0.5421 0.9674 PTAH H&E 0.4240 13 0.1657 0.7903 0.1488 PTAH MT 0.5446 13 0.0091 0.8427 0.0543 PTAH PAS 0.0723 13 0.4986 0.5994 0.8145 SGM AB 0.1653 13 0.6565 0.4244 0.5895 SGM H&E 0.2169 13 0.6859 0.3794 0.4765 SGM MT 0.0537 13 0.5125 0.5873 0.8617 SGM PAS 0.3414 13 0.2582 0.7511 0.2537 SGM PTAH 0.1573 13 0.6518 0.4310 0.6078 VVG AB 0.0575 13 0.5096 0.5898 0.8519 VVG H&E 0.7072 13 0.2559 0.9054 0.0069* VVG MT 0.2495 13 0.3496 0.7037 0.4111 VVG PAS 0.4759 13 0.1018 0.8136 0.1002 VVG PTAH 0.5772 13 0.0384 0.8560 0.0389* VVG SGM 0.1454 13 0.4409 0.6447 0.6355 Graphical Representation Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 F.M Onyije et al / Journal of Cancer Research and Practice xxx (2016) 1e9 References Dio K Computer-aided Diagnosis in Medical Imaging: Historical Review, Current Status and Future Potential vol 31 2007:198e211 (4e5) Mendez AJ, Tahoces PG, Lado MJ, Souto M, Vidal JJ Computer-aided diagnosis: automatic detection of malignant masses in digitized mammograms Med Phys 1998 Jun;25:957e964 Doyle S, Feldman M, Tomaszewski J, Madabhushi A A boosted bayesian multiresolution classifier for prostate cancer detection from digitized needle biopsies IEEE Trans Biomed Eng 2012;59(5):1205e1218 http://dx.doi.org/ 10.1109/TBME.2010.2053540 (Epub 2010 Jun 21) Gurcan MN, Boucheron L, Can A, Madabhushi A, Raipoot, Yener B Histopathological image analysis: a review IEEE Rev Biomed Eng 2009;2:147e171 Basavanhally AN, Ganesan S, Agner S, et al Computerized image-based detection and grading of lymphocytic inltration in HER2ỵ breast cancer histopathology IEEE Trans Biomed Eng 2010 Mar;57(3):642e653 Murphy DB Fundamentals of Light Microscopy and Electronic Imaging WileyLiss; 2001 Schneider CA, Rasband WS, Eliceiri KW NIH Image to ImageJ: 25 years of image analysis Natl Methods 2012;9(7):671e675 Collins TJ ImageJ for microscopy BioTech 2007;43(1):25e30 Ellen CJ Quantitative analysis of histological staining and fluorescence using imageJ Anat Rec 2013;296(3):378e381 lise da morfometria nuclear: 10 Andrea CE, Bleggi-Torres LF, Alves MTS Ana ~o de imagem Braz J descriỗ~ ao da metodologia e o papel dos softwares de ediỗa Pathol Lab Med 2008;44(1):51e57 11 Rangan GK, Tesch GH Quantification of renal pathology by image analysis (Methods in Renal Research) Nephrology 2007;12(6):553e558 12 Avwioro OG Staining In: Histochemistry and Tissue Pathology Principles and Techniques third ed Claverianum Press Nigeria Limited; 2014:133e168 13 Suvarna SK, Layton C, Bancroft JO Bancroft's Theory and Practice of Histopathological Techniques seventh ed Churchill Livingstone; 2013 14 Harris HF On the rapid conversion of haematoxlin into haematein in staining reactions J Appl Microsc Lab 1900;3:777 15 Masson P Some histological methods Trichrome staining and their preliminary technique Bull Int Assoc Med 1929;2:75 16 McManus JFA, Mowry RW Staining Methods, Histologic and Histochemical London: Harper & Row; 1964:268 17 Shum MW, Hon JKY A modified phosphotugstic acid haematoxylin stain for formalin fixed tissue J Med Lab Technol 1969;26:38 18 Southgate HW Note on preparing mucicarmine J Pathology Bacteriol 1927;30: 729 19 Mowry RW Alcian blue technique for the histochemical study of acid carbohydrates J Histochem Cytochem 1956;4:407 20 Verhoef FH Some staining methods of wide applicability, including a rapid differential stain for elastic tissue J Am Med Assoc 1908;50:876 21 Horobin RW Structural staining relationships in histochemistry and Biological Staining I J Microsc 1980;119:345e355 22 Prento P Staining of Macromolecules Biotechnic and Histochemistry vol 84 2009:139e158 23 Horobin RW, Bennion PJ The interrelation of the size and substantivity of dyes: therole of van der waal's attractions and hydrophobic bonding in biological staining Histochromemie 1973;33:191e204 24 Stephen MF, Jagdish B, Michael JS, Douglas W, William CW, Marcos R Pathologic fibrosis and matrix connective tissue in the subaortic myocardium of patients with hypertrophic cardiomyopathy J Am Cell Cardiol 1991;17(6): 1343e1351 25 Street JM, Souza ACP, Alvarez-Prats A, et al Automated quantification of renal fibrosis with Sirius Red and polarization contrast microscopy Physiol Rep 2014;2(7) http://dx.doi.org/10.14814/phy2.12088 26 Kumar V, Abul K, Jon CA Robbins Basic Pathology ninth ed Elsevier Saunders; 2013:689 27 Vanni R Uterus Leiomyoma Atlas Genet Cytogenet Oncol Haematol http:// atlasgeneticsoncology.org/Tumors/leiomyomID5031.html Retrieved 12 of March 2016 28 NewScientist https://books.google.com.ng/books/about/New_Scientist.html? id¼dyM2kv5r7-gC Acccessed 13, March, 2016 29 Ouyang J, Guzman M, Desoto-Lapaix F, Pincus MR, Wieczorek R Utility of desmin and a Masson's trichrome method to detect early acute myocardial infarction in autopsy tissues Int J Clin Exp Pathol 2010;3(1):98e105 Please cite this article in press as: Onyije FM, et al., Computer-assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin-eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016), http:// dx.doi.org/10.1016/j.jcrpr.2016.11.002 ... al., Computer- assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin- eosin with special dyes on tissue, Journal of Cancer... al., Computer- assisted image analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin- eosin with special dyes on tissue, Journal of Cancer... analysis in the diagnosis of gynaecological lesions: A quantitative and comparative investigation of haematoxylin- eosin with special dyes on tissue, Journal of Cancer Research and Practice (2016),