Journal Article
. 2007 Mar; 2:8.
doi: 10.1186/1746-1596-2-8.

Quantitative image analysis of immunohistochemical stains using a CMYK color model

Nhu-An Pham 1 Andrew Morrison  Joerg Schwock  Sarit Aviel-Ronen  Vladimir Iakovlev  Ming-Sound Tsao  James Ho  David W Hedley  
Affiliations
  • PMID: 17326824
  •     22 References
  •     57 citations

Abstract

Background: Computer image analysis techniques have decreased effects of observer biases, and increased the sensitivity and the throughput of immunohistochemistry (IHC) as a tissue-based procedure for the evaluation of diseases.

Methods: We adapted a Cyan/Magenta/Yellow/Key (CMYK) model for automated computer image analysis to quantify IHC stains in hematoxylin counterstained histological sections.

Results: The spectral characteristics of the chromogens AEC, DAB and NovaRed as well as the counterstain hematoxylin were first determined using CMYK, Red/Green/Blue (RGB), normalized RGB and Hue/Saturation/Lightness (HSL) color models. The contrast of chromogen intensities on a 0-255 scale (24-bit image file) as well as compared to the hematoxylin counterstain was greatest using the Yellow channel of a CMYK color model, suggesting an improved sensitivity for IHC evaluation compared to other color models. An increase in activated STAT3 levels due to growth factor stimulation, quantified using the Yellow channel image analysis was associated with an increase detected by Western blotting. Two clinical image data sets were used to compare the Yellow channel automated method with observer-dependent methods. First, a quantification of DAB-labeled carbonic anhydrase IX hypoxia marker in 414 sections obtained from 138 biopsies of cervical carcinoma showed strong association between Yellow channel and positive color selection results. Second, a linear relationship was also demonstrated between Yellow intensity and visual scoring for NovaRed-labeled epidermal growth factor receptor in 256 non-small cell lung cancer biopsies.

Conclusion: The Yellow channel image analysis method based on a CMYK color model is independent of observer biases for threshold and positive color selection, applicable to different chromogens, tolerant of hematoxylin, sensitive to small changes in IHC intensity and is applicable to simple automation procedures. These characteristics are advantageous for both basic as well as clinical research in an unbiased, reproducible and high throughput evaluation of IHC intensity.

Phosphorylated histone H3, Ki-67, p21, fatty acid synthase, and cleaved caspase-3 expression in benign and atypical granular cell tumors.
Payal Kapur, Dinesh Rakheja, +3 authors, Mai P Hoang.
Arch Pathol Lab Med, 2007 Jan 18; 131(1). PMID: 17227124
Carbonic anhydrase IX expression, hypoxia, and prognosis in patients with uterine cervical carcinomas.
David Hedley, Melania Pintilie, +5 authors, Richard Hill.
Clin Cancer Res, 2003 Dec 05; 9(15). PMID: 14654550
Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment I.
R A Walker.
Histopathology, 2006 Sep 19; 49(4). PMID: 16978204
Review.
Interlaboratory comparison of immunohistochemical testing for HER2: results of the 2004 and 2005 College of American Pathologists HER2 Immunohistochemistry Tissue Microarray Survey.
Patrick L Fitzgibbons, Douglas A Murphy, +3 authors, Immunohistochemistry Committee, College of American Pathologists.
Arch Pathol Lab Med, 2006 Nov 09; 130(10). PMID: 17090184
Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis.
H A Lehr, C M van der Loos, P Teeling, A M Gown.
J Histochem Cytochem, 1998 Dec 19; 47(1). PMID: 9857219
Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II.
C R Taylor, R M Levenson.
Histopathology, 2006 Sep 19; 49(4). PMID: 16978205
Highly Cited. Review.
C-erbB-2 immunostaining: problems with interpretation.
E W Kay, C J Walsh, +2 authors, M Leader.
J Clin Pathol, 1994 Sep 01; 47(9). PMID: 7962650    Free PMC article.
p63, cytokeratin 5, and P-cadherin: three molecular markers to distinguish basal phenotype in breast carcinomas.
Irina Matos, Rozany Dufloth, +2 authors, Fernando Schmitt.
Virchows Arch, 2005 Jul 14; 447(4). PMID: 16012853
Comparison of quantification of histochemical staining by hue-saturation-intensity (HSI) transformation and color-deconvolution.
Arnout C Ruifrok, Ruth L Katz, Dennis A Johnston.
Appl Immunohistochem Mol Morphol, 2003 Mar 01; 11(1). PMID: 12610362
Interpretation and quantification of immunostains.
T Seidal, A J Balaton, H Battifora.
Am J Surg Pathol, 2001 Nov 02; 25(9). PMID: 11688582
Review.
Automated selection of DAB-labeled tissue for immunohistochemical quantification.
Eric M Brey, Zahid Lalani, +4 authors, Charles W Patrick.
J Histochem Cytochem, 2003 Apr 22; 51(5). PMID: 12704205
Semiautomated computer-assisted image analysis to quantify 3,3'-diaminobenzidine tetrahydrochloride-immunostained small tissues.
Sandra Leal, Carmen Diniz, +4 authors, Paula Fresco.
Anal Biochem, 2006 Aug 18; 357(1). PMID: 16914112
Measurement of signaling pathway activities in solid tumor fine-needle biopsies by slide-based cytometry.
Jörg Schwock, James C Ho, +2 authors, William R Geddie.
Diagn Mol Pathol, 2007 Aug 28; 16(3). PMID: 17721320
The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice.
Nhu-An Pham, James W Jacobberger, +3 authors, David W Hedley.
Mol Cancer Ther, 2004 Oct 16; 3(10). PMID: 15486191
Digital image analysis for the evaluation of the inflammatory infiltrate in psoriasis.
Amber Y Goedkoop, Menno A de Rie, +5 authors, Maarten C Kraan.
Arch Dermatol Res, 2005 Jul 14; 297(2). PMID: 16012877
Multicolor chromosome painting in diagnostic and research applications.
Sabine Langer, Jürgen Kraus, Isabell Jentsch, Michael R Speicher.
Chromosome Res, 2004 Feb 27; 12(1). PMID: 14984098
Review.
Quantitative immunohistochemistry by measuring cumulative signal strength accurately measures receptor number.
Kristina A Matkowskyj, Randal Cox, Robert T Jensen, Richard V Benya.
J Histochem Cytochem, 2003 Jan 21; 51(2). PMID: 12533529
Quantitative immunohistochemical evaluation of HER2/neu expression with HercepTestTM in breast carcinoma by image analysis.
Y Hatanaka, K Hashizume, +4 authors, Y Tani.
Pathol Int, 2001 Jan 10; 51(1). PMID: 11148461
Immunohistochemical quantitation of androgen receptor expression using color video image analysis.
D Kim, C W Gregory, G J Smith, J L Mohler.
Cytometry, 1999 Nov 30; 35(1). PMID: 10554175
Theory and implementation of an electronic, automated measurement system for images obtained from immunohistochemically stained slides.
Gian Kayser, Dominik Radziszowski, +2 authors, Klaus Kayser.
Anal Quant Cytol Histol, 2006 Mar 29; 28(1). PMID: 16566277
Up-regulation of the redox mediators thioredoxin and apurinic/apyrimidinic excision (APE)/Ref-1 in hypoxic microregions of invasive cervical carcinomas, mapped using multispectral, wide-field fluorescence image analysis.
David Hedley, Melania Pintilie, +6 authors, Richard Hill.
Am J Pathol, 2004 Jan 27; 164(2). PMID: 14742261    Free PMC article.
Methods and biomarkers for the diagnosis and prognosis of cancer and other diseases: towards personalized medicine.
Judy E Anderson, Lise Lotte Hansen, +4 authors, Marek Los.
Drug Resist Updat, 2006 Oct 03; 9(4-5). PMID: 17011811
Review.
Interferon regulatory factor 6 promotes cell cycle arrest and is regulated by the proteasome in a cell cycle-dependent manner.
Caleb M Bailey, Daniel E Abbott, +2 authors, Mary J C Hendrix.
Mol Cell Biol, 2008 Jan 24; 28(7). PMID: 18212048    Free PMC article.
Temporal and spatial expression patterns for the tumor suppressor Maspin and its binding partner interferon regulatory factor 6 during breast development.
Caleb M Bailey, Naira V Margaryan, +4 authors, Mary J C Hendrix.
Dev Growth Differ, 2009 Jun 17; 51(5). PMID: 19527266    Free PMC article.
The human placenta releases substances that drive lung cancer into apoptosis.
Sebastian Marwitz, Tobias Zeiser, +6 authors, Torsten Goldmann.
Diagn Pathol, 2009 Aug 25; 4. PMID: 19698096    Free PMC article.
Immunohistochemistry in diagnostic surgical pathology: contributions of protein life-cycle, use of evidence-based methods and data normalization on interpretation of immunohistochemical stains.
Halliday A Idikio.
Int J Clin Exp Pathol, 2010 Feb 04; 3(2). PMID: 20126585    Free PMC article.
Review.
Developmental programming: effect of prenatal steroid excess on intraovarian components of insulin signaling pathway and related proteins in sheep.
Hugo H Ortega, Florencia Rey, Melisa M L Velazquez, Vasantha Padmanabhan.
Biol Reprod, 2010 Feb 12; 82(6). PMID: 20147730    Free PMC article.
Phase II trial of the histone deacetylase inhibitor belinostat in women with platinum resistant epithelial ovarian cancer and micropapillary (LMP) ovarian tumours.
Helen J Mackay, Hal Hirte, +10 authors, Amit M Oza.
Eur J Cancer, 2010 Mar 23; 46(9). PMID: 20304628    Free PMC article.
A dose-escalation phase I trial of nimotuzumab, an antibody against the epidermal growth factor receptor, in patients with advanced solid malignancies.
Benoit You, Anthony Brade, +7 authors, Eric X Chen.
Invest New Drugs, 2010 May 11; 29(5). PMID: 20454832
ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67.
Vilppu J Tuominen, Sanna Ruotoistenmäki, +2 authors, Jorma Isola.
Breast Cancer Res, 2010 Jul 29; 12(4). PMID: 20663194    Free PMC article.
Highly Cited.
Robust automated tumour segmentation on histological and immunohistochemical tissue images.
Ching-Wei Wang, Dean Fennell, +2 authors, Peter Hamilton.
PLoS One, 2011 Mar 10; 6(2). PMID: 21386898    Free PMC article.
Image analysis tools for evaluation of microscopic views of immunohistochemically stained specimen in medical research-a review.
Keerthana Prasad, Gopalakrishna K Prabhu.
J Med Syst, 2011 May 18; 36(4). PMID: 21584771
Review.
Tyrosine phosphatase SHP-1 is expressed higher in multisystem than in single-system Langerhans cell histiocytosis by immunohistochemistry.
Ichiro Murakami, Takashi Oka, +7 authors, Tadashi Yoshino.
Virchows Arch, 2011 May 24; 459(2). PMID: 21604205
Automation of immunohistochemical evaluation in breast cancer using image analysis.
Keerthana Prasad, Avani Tiwari, +2 authors, Muktha Pai.
World J Clin Oncol, 2011 May 26; 2(4). PMID: 21611095    Free PMC article.
Cellular and molecular responses of the basilar terminus to hemodynamics during intracranial aneurysm initiation in a rabbit model.
John Kolega, Ling Gao, +4 authors, Hui Meng.
J Vasc Res, 2011 Jun 01; 48(5). PMID: 21625176    Free PMC article.
Quantitative analysis of p53 expression in human normal and cancer tissue microarray with global normalization method.
Halliday A Idikio.
Int J Clin Exp Pathol, 2011 Jul 09; 4(5). PMID: 21738821    Free PMC article.
Phosphorylation of signal transducer and activator of transcription 3 (STAT3) correlates with Her-2 status, carbonic anhydrase 9 expression and prognosis in esophageal cancer.
Sebastian F Schoppmann, Bettina Jesch, +3 authors, Peter Birner.
Clin Exp Metastasis, 2012 Apr 10; 29(6). PMID: 22484976
Citrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivo.
Bashir M Mohamed, Navin K Verma, +12 authors, Yuri Volkov.
Nanomedicine (Lond), 2012 May 26; 7(8). PMID: 22625207    Free PMC article.
Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis.
Annie Lauzier, Martine Charbonneau, +2 authors, Claire M Dubois.
Arthritis Res Ther, 2012 Jul 06; 14(4). PMID: 22762273    Free PMC article.
Role of proprotein convertases in prostate cancer progression.
Frédéric Couture, François D'Anjou, +2 authors, Robert Day.
Neoplasia, 2012 Dec 12; 14(11). PMID: 23226097    Free PMC article.
Identifying survival associated morphological features of triple negative breast cancer using multiple datasets.
Chao Wang, Thierry Pécot, +3 authors, Kun Huang.
J Am Med Inform Assoc, 2013 Apr 16; 20(4). PMID: 23585272    Free PMC article.
TOSO promotes β-cell proliferation and protects from apoptosis.
G Dharmadhikari, M Mühle, +5 authors, K Maedler.
Mol Metab, 2012 Jan 01; 1(1-2). PMID: 24024120    Free PMC article.
Rate of atherosclerosis progression in ApoE-/- mice long after discontinuation of cola beverage drinking.
Matilde Otero-Losada, Gabriel Cao, +3 authors, José Milei.
PLoS One, 2014 Mar 29; 9(3). PMID: 24670925    Free PMC article.
Secretion of von Willebrand factor by endothelial cells links sodium to hypercoagulability and thrombosis.
Natalia I Dmitrieva, Maurice B Burg.
Proc Natl Acad Sci U S A, 2014 Apr 16; 111(17). PMID: 24733925    Free PMC article.
Stromal expression of carbonic anhydrase IX in esophageal cancer.
G Jomrich, B Jesch, +4 authors, S F Schoppmann.
Clin Transl Oncol, 2014 Apr 17; 16(11). PMID: 24737069
IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples.
Frency Varghese, Amirali B Bukhari, Renu Malhotra, Abhijit De.
PLoS One, 2014 May 08; 9(5). PMID: 24802416    Free PMC article.
Highly Cited.
Implications of Proprotein Convertases in Ovarian Cancer Cell Proliferation and Tumor Progression: Insights for PACE4 as a Therapeutic Target.
Rémi Longuespée, Frédéric Couture, +8 authors, Robert Day.
Transl Oncol, 2014 May 14;. PMID: 24818756    Free PMC article.
EGF up-regulates miR-31 through the C/EBPβ signal cascade in oral carcinoma.
Wen-Cheng Lu, Shou-Yen Kao, +4 authors, Shu-Chun Lin.
PLoS One, 2014 Sep 18; 9(9). PMID: 25229239    Free PMC article.
Periadventitial atRA citrate-based polyester membranes reduce neointimal hyperplasia and restenosis after carotid injury in rats.
Elaine K Gregory, Antonio R Webb, +3 authors, Melina R Kibbe.
Am J Physiol Heart Circ Physiol, 2014 Sep 23; 307(10). PMID: 25239800    Free PMC article.
Computerized delineation of nuclei in liquid-based pap smears stained with immunohistochemical biomarkers.
Yi Qin, Ann E Walts, Beatrice S Knudsen, Arkadiusz Gertych.
Cytometry B Clin Cytom, 2014 Oct 04; 88(2). PMID: 25280117    Free PMC article.
Staining correction in digital pathology by utilizing a dye amount table.
Pinky A Bautista, Yukako Yagi.
J Digit Imaging, 2015 Jan 07; 28(3). PMID: 25561073    Free PMC article.
Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system.
Aida Laurinaviciene, Benoit Plancoulaine, +7 authors, Arvydas Laurinavicius.
Diagn Pathol, 2015 Jan 08; 9 Suppl 1. PMID: 25565007    Free PMC article.
Effect of low-level laser therapy on bone repair: a randomized controlled experimental study.
Valéria Regina Gonzalez Sella, Fernando Russo Costa do Bomfim, +3 authors, Helio Plapler.
Lasers Med Sci, 2015 Jan 19; 30(3). PMID: 25596935
PACE4 inhibitors and their peptidomimetic analogs block prostate cancer tumor progression through quiescence induction, increased apoptosis and impaired neovascularisation.
Christine Levesque, Frédéric Couture, +4 authors, Robert Day.
Oncotarget, 2015 Feb 17; 6(6). PMID: 25682874    Free PMC article.
Digital separation of diaminobenzidine-stained tissues via an automatic color-filtering for immunohistochemical quantification.
Rong Fu, Xiaomian Ma, Zhaoying Bian, Jianhua Ma.
Biomed Opt Express, 2015 Mar 18; 6(2). PMID: 25780744    Free PMC article.
Elevated sodium and dehydration stimulate inflammatory signaling in endothelial cells and promote atherosclerosis.
Natalia I Dmitrieva, Maurice B Burg.
PLoS One, 2015 Jun 05; 10(6). PMID: 26042828    Free PMC article.
A Model based Survey of Colour Deconvolution in Diagnostic Brightfield Microscopy: Error Estimation and Spectral Consideration.
Peter Haub, Tobias Meckel.
Sci Rep, 2015 Aug 01; 5. PMID: 26223691    Free PMC article.
IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion.
Chen-Yeh Ke, Wen-Lin Xiao, +2 authors, Fen-Hwa Wong.
Sci Rep, 2015 Aug 05; 5. PMID: 26240017    Free PMC article.
Increased SYK activity is associated with unfavorable outcome among patients with acute myeloid leukemia.
Katalin Boros, Alexandre Puissant, +7 authors, Scott J Rodig.
Oncotarget, 2015 Sep 01; 6(28). PMID: 26315286    Free PMC article.
Influence of Texture and Colour in Breast TMA Classification.
M Milagro Fernández-Carrobles, Gloria Bueno, +3 authors, Lucía González-López.
PLoS One, 2015 Oct 30; 10(10). PMID: 26513238    Free PMC article.
A robust nonlinear tissue-component discrimination method for computational pathology.
Jacob S Sarnecki, Kathleen H Burns, +4 authors, Pei-Hsun Wu.
Lab Invest, 2016 Jan 19; 96(4). PMID: 26779829    Free PMC article.
Statistical colour models: an automated digital image analysis method for quantification of histological biomarkers.
Jie Shu, G E Dolman, +2 authors, Mohammad Ilyas.
Biomed Eng Online, 2016 Apr 29; 15. PMID: 27121383    Free PMC article.
Stimulation of triple negative breast cancer cell migration and metastases formation is prevented by chloroquine in a pre-irradiated mouse model.
Gina Bouchard, Hélène Therriault, +4 authors, Benoit Paquette.
BMC Cancer, 2016 Jun 11; 16. PMID: 27282478    Free PMC article.
Immunohistochemical expression of intrarenal renin angiotensin system components in response to tempol in rats fed a high salt diet.
Gabriel Cao, Silvana Lorena Della Penna, +5 authors, María Inés Rosón.
World J Nephrol, 2017 Jan 20; 6(1). PMID: 28101449    Free PMC article.
Siglec-7 restores β-cell function and survival and reduces inflammation in pancreatic islets from patients with diabetes.
Gitanjali Dharmadhikari, Katharina Stolz, +5 authors, Kathrin Maedler.
Sci Rep, 2017 Apr 06; 7. PMID: 28378743    Free PMC article.
Inhibition of ROCK1 kinase modulates both tumor cells and stromal fibroblasts in pancreatic cancer.
Clifford J Whatcott, Serina Ng, +3 authors, Haiyong Han.
PLoS One, 2017 Aug 26; 12(8). PMID: 28841710    Free PMC article.
MK2 and ETV1 Are Prognostic Factors in Esophageal Adenocarcinomas.
Gerd Jomrich, Florian Maroske, +6 authors, Sebastian Friedrich Schoppmann.
J Cancer, 2018 Feb 28; 9(3). PMID: 29483950    Free PMC article.
Tau-tubulin kinase 1 and amyloid-β peptide induce phosphorylation of collapsin response mediator protein-2 and enhance neurite degeneration in Alzheimer disease mouse models.
Seiko Ikezu, Kaitlin L Ingraham Dixie, +3 authors, Tsuneya Ikezu.
Acta Neuropathol Commun, 2020 Feb 06; 8(1). PMID: 32019603    Free PMC article.
PrPC knockdown by liposome-siRNA-peptide complexes (LSPCs) prolongs survival and normal behavior of prion-infected mice immunotolerant to treatment.
Heather Bender, Noelle Noyes, +7 authors, Mark Zabel.
PLoS One, 2019 Jul 23; 14(7). PMID: 31329627    Free PMC article.
Real-time Colorimetric Quantitative Molecular Detection of Infectious Diseases on Smartphone-based Diagnostic Platform.
Kun Yin, Vikram Pandian, +4 authors, Changchun Liu.
Sci Rep, 2020 Jun 04; 10(1). PMID: 32488061    Free PMC article.
Isolation, Characterization and Proliferation of Cancer Cells from Breast Cancer Patients.
Wahyu Widowati, Yusuf Heriady, +9 authors, Mawar Subangkit.
Acta Inform Med, 2019 Jan 30; 26(4). PMID: 30692706    Free PMC article.
Characterization of weaning-induced breast involution in women: implications for young women's breast cancer.
Sonali Jindal, Jayasri Narasimhan, Virginia F Borges, Pepper Schedin.
NPJ Breast Cancer, 2020 Oct 22; 6. PMID: 33083533    Free PMC article.
Structural changes in endocrine pancreas of male Wistar rats due to chronic cola drink consumption. Role of PDX-1.
Gabriel Cao, Julián González, +6 authors, José Milei.
PLoS One, 2021 Jun 12; 16(6). PMID: 34115756    Free PMC article.
Super-Resolution Ultrasound Localization Microscopy on a Rabbit Liver VX2 Tumor Model: An Initial Feasibility Study.
Wei Zhang, Matthew R Lowerison, +3 authors, Pengfei Song.
Ultrasound Med Biol, 2021 May 29; 47(8). PMID: 34045095    Free PMC article.
HER2 Molecular Marker Scoring Using Transfer Learning and Decision Level Fusion.
Suman Tewary, Sudipta Mukhopadhyay.
J Digit Imaging, 2021 Mar 21; 34(3). PMID: 33742331    Free PMC article.
Tumor Temporal Proteome Profiling Reveals the Immunological Triple Offensive Induced by Synthetic Anti-Cancer Salmonella.
Shuxin Yang, Wenjuan Zhao, +10 authors, Nan Li.
Front Immunol, 2021 Sep 08; 12. PMID: 34489962    Free PMC article.
Serum Amyloid A Promotes Inflammation-Associated Damage and Tumorigenesis in a Mouse Model of Colitis-Associated Cancer.
Tanja A Davis, Daleen Conradie, +3 authors, Willem J S de Villiers.
Cell Mol Gastroenterol Hepatol, 2021 Jul 05; 12(4). PMID: 34217896    Free PMC article.
Characterization of the tumor immune microenvironment of sinonasal squamous-cell carcinoma.
Jeffrey T Gu, Natalie Claudio, +3 authors, Ferdinando Pucci.
Int Forum Allergy Rhinol, 2021 Sep 13; 12(1). PMID: 34510766    Free PMC article.
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IEEE Trans Biomed Eng, 2021 Oct 12; 69(4). PMID: 34633926    Free PMC article.