Peter Ella-Tongwiis1,2,3, Alexander Makanga2,4, Iqbal Shergill1,2,3,5, Stephen Fôn Hughes2,3,6. 1. North Wales and North West Urological Research Centre, Betsi Cadwaladr University Health Board (BCUHB) Wrexham Maelor Hospital, Wrexham, Wales, UK. 2. North Wales Clinical Research Centre, Betsi Cadwaladr University Health Board (BCUHB) Wrexham Maelor Hospital, Wrexham, Wales, UK. 3. Faculty of Social and Life Sciences, Wrexham Glyndwr University, Wrexham, UK. 4. Department of Histopathology, BCUHB Glan Clwyd Hospital, Denbighshire, Wales, UK. 5. The Alan de Bolla Department of Urology, BCUHB Wrexham Maelor Hospital, Wrexham, Wales, UK. 6. North Wales and North West Urological Research Centre, Betsi Cadwaladr University Health Board (BCUHB) Wrexham Maelor Hospital, Wrexham, Wales, UK. Stephen.hughes6@wales.nhs.uk.
Abstract
BACKGROUND: Immunohistochemistry (IHC) has become a valuable laboratory technique for diagnosing, evaluating metastasis and informing treatment selection in several cancers. Standardization however remains a limiting factor in IHC. The main aim of this research study was to optimise, validate and standardize antibodies and IHC protocols for cancer research. METHODS: Seven monoclonal mouse and rabbit antibodies were optimised using formalin-fixed paraffin embedded (FFPE) human tissue blocks. 4um sections of FFPE block were stained using the Roche Ventana XT or Ventana ULTRA IHC automated analysers. This study modified manufacturer recommended protocols by using a unique antigen retrieval method, adding an amplification step, varying primary antibody incubation times, as well as using the Roche Ventana Ultraview detection system. RESULTS: Optimum antibody localisation was observed in modified IHC protocols in comparison with manufacturer recommended protocols for anti-CEACAM-1, anti-CD31, anti-COX-2, anti-HER-2/neu, anti-S100P, anti-thrombomodulin and anti-VEGFR-3. Majority of antibodies required more than one modification of the initial protocol. For anti-VEGFR-3 optimum staining was observed following 4 protocol modifications. CONCLUSIONS: This study has optimised and standardized several tissue-based biomarkers that may be, in the future, used to screen, diagnose and monitor patients with certain cancer, such as bladder cancer. Accurate data on optimised protocols reduce time and resources wasted on experimental protocols, and ultimately help identify biomarkers or biomarker panels, which may be used to select treatment regimens for various cancers.
BACKGROUND: Immunohistochemistry (IHC) has become a valuable laboratory technique for diagnosing, evaluating metastasis and informing treatment selection in several cancers. Standardization however remains a limiting factor in IHC. The main aim of this research study was to optimise, validate and standardize antibodies and IHC protocols for cancer research. METHODS: Seven monoclonal mouse and rabbit antibodies were optimised using formalin-fixed paraffin embedded (FFPE) human tissue blocks. 4um sections of FFPE block were stained using the Roche Ventana XT or Ventana ULTRA IHC automated analysers. This study modified manufacturer recommended protocols by using a unique antigen retrieval method, adding an amplification step, varying primary antibody incubation times, as well as using the Roche Ventana Ultraview detection system. RESULTS: Optimum antibody localisation was observed in modified IHC protocols in comparison with manufacturer recommended protocols for anti-CEACAM-1, anti-CD31, anti-COX-2, anti-HER-2/neu, anti-S100P, anti-thrombomodulin and anti-VEGFR-3. Majority of antibodies required more than one modification of the initial protocol. For anti-VEGFR-3 optimum staining was observed following 4 protocol modifications. CONCLUSIONS: This study has optimised and standardized several tissue-based biomarkers that may be, in the future, used to screen, diagnose and monitor patients with certain cancer, such as bladder cancer. Accurate data on optimised protocols reduce time and resources wasted on experimental protocols, and ultimately help identify biomarkers or biomarker panels, which may be used to select treatment regimens for various cancers.
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