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Application of Photoshop-based Image Analysis to Quantification of Hormone Receptor Expression in Breast Cancer

Application of Photoshop-based Image Analysis to Quantification of Hormone Receptor Expression in Breast Cancer,Hans-Anton Lehr,David A. Mankoff,David

Application of Photoshop-based Image Analysis to Quantification of Hormone Receptor Expression in Breast Cancer   (Citations: 125)
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SUMMARY The benefit of quantifying estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer is well established. However, in routine breast cancer diag- nosis, receptor expression is often quantified in arbitrary scores with high inter- and in- traobserver variability. In this study we tested the validity of an image analysis system em- ploying inexpensive, commercially available computer software on a personal computer. In a series of 28 invasive ductal breast cancers, immunohistochemical determinations of ER and PR were performed, along with biochemical analyses on fresh tumor homogenates, by the dextran-coated charcoal technique (DCC) and by enzyme immunoassay (EIA). From each immunohistochemical slide, three representative tumor fields ( 3 20 objective) were captured and digitized with a Macintosh personal computer. Using the tools of Photoshop software, optical density plots of tumor cell nuclei were generated and, after background subtraction, were used as an index of immunostaining intensity. This immunostaining index showed a strong semilogarithmic correlation with biochemical receptor assessments of ER (DCC, r 5 0.70, p , 0.001; EIA, r 5 0.76, p , 0.001) and even better of PR (DCC, r 5 0.86; p , 0.01; EIA, r 5 0.80, p , 0.001). A strong linear correlation of ER and PR quantification was also seen between DCC and EIA techniques (ER, r 5 0.62, p , 0.001; PR, r 5 0.92, p , 0.001). This study demonstrates that a simple, inexpensive, commercially available software pro- gram can be accurately applied to the quantification of immunohistochemical hormone re- ceptor studies. (J Histochem Cytochem 45:1559-1565, 1997)
Published in 1997.
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    • ...The interpretation of immunostains has made a rapid development from manual counting to fully automated techniques for image capture and analysis [1, 11, 13]...
    • ...The reports on quantitative automated image analysis focus on various parameters, like vascular density [14], nuclear staining or intensity of staining [11, 15]...

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    • ... OPN abundance was quantified using Adobe Photoshop Software ...

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    • ...This step is critical for vein grafts to generate stable contractions and relaxations comparable to the physiological situation. As the luminal diameter and wall thickness of vein grafts varies, the actual circumferential tensions on individual vein graft rings are different for comparable intra-luminal pressures. Contractile function was tested using 3 constrictors added in the following sequence: thromboxane mimetic U46619 (9,11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α, 3 × 10–7M), phenylephrine (1 × 10–6M), and KCl (60 mM). In all cases, the vessels were washed at least 3 times to return them to baseline tension prior to addition of the next constrictor. The constrictors were used at high concentrations to test the maximum contractions in vessel rings. To study vascular relaxant properties, preparations were pre-contracted to U46619 (3 × 10–8M), which induces a submaximal contraction suitable for vessel relaxation experiments. Maximal endothelium-dependent relaxation was then induced by carbachol (1 × 10–5M), and endothelial-independent relaxation by spermine-NONOate (SPNO, an NO donor, 1 × 10–5M). The procedure was repeated after a 30-min incubation with the NOS inhibitor L-NAME (1 × 10–4M) or with the iNOS inhibitor 1400W (1 × 10–5M).Histology and ImmunohistochemistryVein grafts and carotid arteries were fixed overnight in formalin, embedded in paraffin and cut into 4-μm sections. Haematoxylin and eosin (HE) staining was applied for morphology and planimetry studies. The area of neointima and neoadventitia were determined using a computerized image-analysis system (Image Pro Plus). The neointima and neoadventitia are defined as the newly formed cell layers on the luminal side and the adventitial side, respectively, of the original vein remnant. As the original vein is only formed by 2–3 layers of cells, no obvious media layer can be defined in this model. In the first week, the original vein remnant appears as a red line with HE staining, which indicates the interface of the neointima and neoadventitia (fig. 1a, day 7). At 2–4 weeks, the original vein is not easily identified, while the neointima and neoadventitia are easily split along the original vein and represent different morphological appearance. The neoadventitia is usually a solid tissue layer with much higher cell density than normal connective tissue. In well-adapted vein grafts, there might be healthy connective tissue surrounding the neoadventitia with a clear interface. In some cases, neoadventitia was found to expand to adjacent muscles, having no connective tissue in between.
      1Fig. 1. Dynamics of re-endothelialization and vascular remodelling in vein grafts up to 4 weeks. a HE staining. Acute inflammation at day 7 showing copious infiltrated leukocyte (indicated by arrow). Cells accumulated on both intimal and adventitial sides to form the neointima and neoadventitia. At 14 days a complete layer of endothelial-like cells was present (indicated by arrows). The open arrowheads in a indicate the interface between neointima and neoadventitia. b eNOS staining in contralateral carotid artery and vein grafts at 14 and 28 days. Carotid arteries and vein grafts at 28 days only showed specific eNOS expression localized in the endothelium as indicated by arrows. c Planimetric analysis of graft neointimal and neoadventitial areas. d Intensity of eNOS staining in the re-endothelialized graft at 28 days versus carotid artery. ** p < 0.01; + p < 0.01, compared with days 1–3 in c and compared with carotid artery in d (n = 5–8).F01
      For immunohistochemistry, slides were de-waxed, pressure cooked for antigen retrieval, then treated with the appropriate primary antibody followed by a complementary secondary antibody and the peroxidase/DAB chromophore. The antibodies used were: eNOS (610298; BD Transduction Laboratories; 1:100 dilution), iNOS (ab15323; Abcam; 1:50 dilution), von Willebrand factor (vWF, A0082; Dako; 1:2,000 dilution), Ki67 (ab15580; Abcam; 1:100 dilution), CD14 (ab25092; Abcam; 1:4,000). The expression level of proteins was quantified using Image-pro Plus by colour matching a stained region, then creating an image mask followed by determination of the total stained area and of the average absorbed pixel intensity [...

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