Eric Deutsch for their role in developing the MISFISHIE specification, discussions over many years with Dr

Eric Deutsch for their role in developing the MISFISHIE specification, discussions over many years with Dr. in tissue, particularly in small biopsy samples. Experimental Design; Biomaterials and Treatment, i.e. tissue processing, fixation medium, storage conditions; Reporters, i.e. antibody source, clone, concentration; Staining, i.e. antigen retrieval method, staining protocol; Imaging Data, i.e. images of immunostains; Image Characterization, i.e. algorithm for reporting and analyzing the immunostaining images, criteria for positivity By applying MISFISHIE rules to experiments all potential sources of variance in tissue based experiments will, in principle, be revealed. Although documenting all steps of immunohistochemical experiments provides a basis for targeting those parts of the procedure that are the source of greatest variance, implementation of quality control procedures is the next step. Using on slide positive and negative control tissues (Mengel et al. 2005) and parallel immunostains of tissue microarrays that contain a variety of control tissues (Hsu et al. 2002) are ways to increase confidence that an immunohistochemical finding is accurate. Standardization Standardization of procedures in different labs would increase the reliability of an immunohistochemical procedure. A step in addressing standardization of tissue processing is to document how different labs process their samples. A component of an ongoing study by the NIH-funded Prostate Cancer SPOREs was documenting the timing for different steps of tissue processing at the SPORE sites. Data recorded by the tissue processors illustrate the range in time for processing tissue from ethanol into paraffin0 to 310?min (Fig.?3). This sequence is but one set of intermediate steps in the tissue processing cycle. The effect of this range of processing time on antigen immunoreactivity is being assessed. Unfortunately, documentation of Trofosfamide the time required for the entire sequence of tissue processing is virtually never done due to the imprecision and impracticality of being able to accurately record the time required for many steps, such as time the tissue sits at ambient temperature on the bench before fixation and time in formalin before initiating tissue processing. Open in a separate window Fig.?3 Time to process blocks of Trofosfamide formaldehyde-fixed, tissue by tissue processors at seven different institutions (NIH-funded Prostate Cancer SPOREs) from the first ethanol dehydration step through paraffin infiltration (Fine SW, Trock B, Reuter VE, Ayala G, Cheville JC, Fearn P, Jenkins RB, Knudsen BS, Loda M, Netto GJ, Said J, Shah RB, Simko J, Troncoso P, True LD, Yang XJ, Rubin MA, DeMarzo AM (2007) Effects of tissue processing on biomarker analysis in prostate needle biopsies: a multi-institutional study. Annual Meeting of USCCanadian Academy of Pathology Although desirable, standardization of routine tissue handling procedures and of tissue localization methods does not appear imminent due to clinical and practical considerations, which differ by laboratory, and the absence of compelling evidence that such standardization will be of value. What would be of value would be developing a metric to assess the quality of preservation of macromolecules Trofosfamide in fixed tissue. Methods are being applied that measure integrity of nucleic acids in fixed tissue (Jewell et al. 2002). Although assessment of protein integrity is a greater challenge, mass spectrometry is being used as a protein integrity assessment tool (Shi et al. 2006). Quantification by immunoperoxidase histochemistry The conditions necessary to ensure that tissue quality is sufficient to minimize the possibility of false negative and false positive immunohistochemical results pale at the challenge posed by the goal of obtaining reliable and reproducible quantifications of molecules in sections of tissue. To truly quantify levels of gene expression by immunohistochemistry entails developing a standard curve for each antigen and each immunohistochemical method. Based on a standard curve, where known (either absolute or relative) numbers of molecules per unit of tissue (or, ideally, per average cell), is determined at multiple concentrations over the range of anticipated expression level in the tissue of interest, the expression level of the molecule of interest can be calculated. However, with only rare exceptions, expression levels of Rabbit Polyclonal to CNTROB gene products are quantified by immunohistochemical methods and published without reference to a standard curve for that gene produce in that.