• Clinical quantitation of immune signature in follicular lymphoma by RT-PCR-based gene expression profiling.

      Byers, Richard J; Sakhinia, Ebrahim; Joseph, Preethi; Glennie, Caroline; Hoyland, Judith A; Menasce, Lia P; Radford, John A; Illidge, Timothy M; Department of Histopathology and Manchester Molecular Diagnostic Center, Manchester Royal Infirmary, Central Manchester, UK. r.byers@manchester.ac.uk (2008-05-01)
      Microarray gene expression profiling studies have demonstrated immune response gene signatures that appear predictive of outcome in follicular lymphoma (FL). However, measurement of these marker genes in routine practice remains difficult. We have therefore investigated the immune response in FL using real-time polymerase chain reaction (PCR) to measure expression levels of 35 candidate Indicator genes, selected from microarray studies, to polyA cDNAs prepared from 60 archived human frozen lymph nodes, in parallel with immunohistochemical analysis for CD3, CD4, CD7, CD8, CD10, CD20, CD21, and CD68. High levels of CCR1, a marker of monocyte activation, were associated with a shorter survival interval, and high levels of CD3 with better survival, while immunohistochemistry demonstrated association of high numbers of CD68(+) macrophages with a shorter survival interval and of high numbers of CD7(+) T cells with a longer survival interval. The results confirm the role of the host immune response in outcome in FL and identify CCR1 as a prognostic indicator and marker of an immune switch between macrophages and a T cell-dominant response. They demonstrate the utility of polyA DNA and real-time PCR for measurement of gene signatures and the applicability of using this type of "molecular block" in clinical practice.
    • Quantitative multiplexed quantum dot immunohistochemistry.

      Sweeney, Elizabeth; Ward, Timothy H; Gray, N; Womack, C; Jayson, Gordon C; Hughes, Andrew; Dive, Caroline; Byers, Richard J; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, 420 4BX, UK. (2008-09-19)
      Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.
    • Quantum dots brighten biological imaging.

      Byers, Richard J; Hitchman, Elizabeth R; School of Cancer and Enabling Sciences, University of Manchester, Manchester, UK. richard.byers@cmft.nhs.uk (2011-02)
      Quantum dots (QDs) are novel photostable semiconductor nanocrystals possessing wide excitation spectra and narrow, symmetrical emission spectra and can be conjugated to a wide range of biological targets, including proteins, antibodies and nucleic acid probes. These characteristics have provoked considerable interest in their use for bioimaging. Much investigation has been performed into their use for multiplex immunohistochemistry and in situ hybridisation which, when combined with multispectral imaging, has enabled quantitation and colocalisation of gene expression in clinical tissue. Many advances have recently been made using QDs for live cell and in vivo imaging, in which QD-labelled molecules can be tracked and visualised in 3-D. This review aims to outline the beneficial properties presented by QDs along with important advances in their biological application.
    • Quantum dots light up pathology.

      Tholouli, E; Sweeney, Elizabeth; Barrow, E; Clay, V; Hoyland, Judith A; Byers, Richard J; Department of Clinical Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, UK. (2008-11)
      Quantum dots (QDs) are novel nanocrystal fluorophores with extremely high fluorescence efficiency and minimal photobleaching. They also possess a constant excitation wavelength together with sharp and symmetrical tunable emission spectra. These unique optical properties make them near-perfect fluorescent markers and there has recently been rapid development of their use for bioimaging. QDs can be conjugated to a wide range of biological targets, including proteins, antibodies, and nucleic acid probes, rendering them of particular interest to pathology researchers. They have been used in multiplex immunohistochemistry and in situ hybridization, which when combined with multispectral imaging, has enabled quantitative measurement of gene expression in situ. QDs have also been used for live in vivo animal imaging and are now being applied to an ever-increasing range of biological problems. These are detailed in this review, which also acts to outline the important advances that have been made in their range of applications. The relative novelty of QDs can present problems in their practical use and guidelines for their application are given.