Quantitative image analysis of immunohistochemical stains using a CMYK color model
Tóm tắt
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.
We adapted a Cyan/Magenta/Yellow/Key (CMYK) model for automated computer image analysis to quantify IHC stains in hematoxylin counterstained histological sections.
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.
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.
Từ khóa
Tài liệu tham khảo
Anderson JE, Hansen LL, Mooren FC, Post M, Hug H, Zuse A, Los M: Methods and biomarkers for the diagnosis and prognosis of cancer and other diseases: towards personalized medicine. Drug Resist Updat. 2006, 9 (4-5): 198-210. 10.1016/j.drup.2006.08.001.
Walker RA: Quantification of immunohistochemistry-issues concerning methods, utility and semiquantitative assessment I. Histopathology. 2006, 49 (4): 406-410. 10.1111/j.1365-2559.2006.02514.x.
Hatanaka Y, Hashizume K, Kamihara Y, Itoh H, Tsuda H, Osamura RY, Tani Y: Quantitative immunohistochemical evaluation of HER2/neu expression with HercepTestTM in breast carcinoma by image analysis. Pathol Int. 2001, 51 (1): 33-36. 10.1046/j.1440-1827.2001.01162.x.
Langer S, Kraus J, Jentsch I, Speicher MR: Multicolor chromosome painting in diagnostic and research applications. Chromosome Res. 2004, 12 (1): 15-23. 10.1023/B:CHRO.0000009326.21752.88.
Kayser G, Radziszowski D, Bzdyl P, Sommer R, Kayser K: Theory and implementation of an electronic, automated measurement system for images obtained from immunohistochemically stained slides. Anal Quant Cytol Histol. 2006, 28 (1): 27-38.
Brey EM, Lalani Z, Johnston C, Wong M, McIntire LV, Duke PJ, Patrick CW: Automated selection of DAB-labeled tissue for immunohistochemical quantification. J Histochem Cytochem. 2003, 51 (5): 575-584.
Goedkoop AY, de Rie MA, Teunissen MB, Picavet DI, van der Hall PO, Bos JD, Tak PP, Kraan MC: Digital image analysis for the evaluation of the inflammatory infiltrate in psoriasis. Arch Dermatol Res. 2005, 297 (2): 51-59. 10.1007/s00403-005-0578-4.
Kim D, Gregory CW, Smith GJ, Mohler JL: Immunohistochemical quantitation of androgen receptor expression using color video image analysis. Cytometry. 1999, 35 (1): 2-10. 10.1002/(SICI)1097-0320(19990101)35:1<2::AID-CYTO2>3.0.CO;2-Y.
Matkowskyj KA, Cox R, Jensen RT, Benya RV: Quantitative immunohistochemistry by measuring cumulative signal strength accurately measures receptor number. J Histochem Cytochem. 2003, 51 (2): 205-214.
Ruifrok AC, Katz RL, Johnston DA: Comparison of quantification of histochemical staining by hue-saturation-intensity (HSI) transformation and color-deconvolution. Appl Immunohistochem Mol Morphol. 2003, 11 (1): 85-91. 10.1097/00022744-200303000-00014.
Taylor CR, Levenson RM: Quantification of immunohistochemistry-issues concerning methods, utility and semiquantitative assessment II. Histopathology. 2006, 49 (4): 411-424. 10.1111/j.1365-2559.2006.02513.x.
Leal S, Diniz C, Sa C, Goncalves J, Soares AS, Rocha-Pereira C, Fresco P: Semiautomated computer-assisted image analysis to quantify 3,3'-diaminobenzidine tetrahydrochloride-immunostained small tissues. Anal Biochem. 2006, 357 (1): 137-143. 10.1016/j.ab.2006.07.017.
Lehr HA, van der Loos CM, Teeling P, Gown AM: Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem. 1999, 47 (1): 119-126.
Hedley D, Pintilie M, Woo J, Nicklee T, Morrison A, Birle D, Fyles A, Milosevic M, Hill R: 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. Am J Pathol. 2004, 164 (2): 557-565.
Hedley D, Pintilie M, Woo J, Morrison A, Birle D, Fyles A, Milosevic M, Hill R: Carbonic anhydrase IX expression, hypoxia, and prognosis in patients with uterine cervical carcinomas. Clin Cancer Res. 2003, 9 (15): 5666-5674.
Schwock J, Ho JC, Luther E, Hedley D, Geddie WR: Detection and measurement of phosphorylated signaling proteins in solid tumor fine-needle biopsies by laser-scanning cytometry. Diagostic Molecular Pathology.
Pham NA, Jacobberger JW, Schimmer AD, Cao P, Gronda M, Hedley DW: 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. Mol Cancer Ther. 2004, 3 (10): 1239-1248.
Iakovlev V, Morrison A, Pintile M, Hill R, Hedley D: Quantitative assessment of heterogeneously expressed markers within histological sections. 57th Annual Meeting of the Canadian Association of Pathologists 2006, St. John, NL, Canada. 2006
Aviel-Ronen S, Ling C, Lu M, Lau D, Ho JC, Liu N, Tsao MS: p16 methylation and protein expression in non-small cell lung cancer: September 16-21 2006; Montreal, Quebec Canada.2006, , Modern Pathology 19: supplement 3:
Seidal T, Balaton AJ, Battifora H: Interpretation and quantification of immunostains. Am J Surg Pathol. 2001, 25 (9): 1204-1207. 10.1097/00000478-200109000-00013.
Kapur P, Rakheja D, Balani JP, Roy LC, Amirkhan RH, Hoang MP: Phosphorylated histone H3, Ki-67, p21, fatty acid synthase, and cleaved caspase-3 expression in benign and atypical granular cell tumors. Arch Pathol Lab Med. 2007, 131 (1): 57-64.
Matos I, Dufloth R, Alvarenga M, Zeferino LC, Schmitt F: p63, cytokeratin 5, and P-cadherin: three molecular markers to distinguish basal phenotype in breast carcinomas. Virchows Arch. 2005, 447 (4): 688-694. 10.1007/s00428-005-0010-7.
Kay EW, Walsh CJ, Cassidy M, Curran B, Leader M: C-erbB-2 immunostaining: problems with interpretation. J Clin Pathol. 1994, 47 (9): 816-822.