Future Uses of the Department of Defense Joint Pathology Center Biorepository (2012) / Chapter Skim
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2 Determinants of the Research Value of Biospecimens
2 Determinants of the Research Value of Biospecimens
Pages 37-64
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From page 37... ...
Clinical data -- including information about the patient and her or his medical history, physical examination, and diagnostic imaging, such as X-rays, CT scans, and the like -- are also collected to inform evaluations.
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Gross Histology Gross Specimen: Slides Specimen: Fixed Fresh FFPE Tissue Tissue Microarrays Digital Blocks Images Cells Body Fluids FFPE Cell (Cytology) Blocks Patient Cytology Slides Cell Personal Smears Information Clinical Data Diagnosis History and Physical Exam Rx-CT Images FIGURE 2-1Relationship between pathologic materials collected and form in which they are preserved for analysis.
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From page 39... ...
constructed from diag nostic blocks of FFPE tissue to permit simultaneous evaluation of expression of specific pathologic features -- proteins by immunohistochemistry, for example -- in hundreds of individual tissue samples from different patients on a single slide (Rimm et al., 2011; Voduc et al., 2008)
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This section presents a brief summary of the considerations that influence the assessment of fitness for those uses. Educational Uses Educational uses have the lowest bar for molecular quality and physical integrity of material and therefore can, in principle, permit the greatest variety of samples.
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Research Uses Research comprises a broad array of activities and a correspondingly broad array of requirements for pathologic specimens. Case reports and historical studies might need slides alone and be limited only by the condition of the slides, but more extensive studies of the mechanism of disease require the freshest materials possible with cryopreservation, snap freezing in liquid nitrogen, or relatively brief times in fixatives to obtain the best results.
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From page 42... ...
Clinical consultation biorepositories have difficulty in controlling, recording, or assessing the sources of preanalytic variation that may compromise the molecular quality of their collections. Thus, technologic solutions for controlling processing and environmental variation and for assessing the molecular quality of processed or stored specimens have been essential for the continued evolution and usefulness of clinical consultation biorepositories for biomedical research.
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. · Information technology solutions that allow annotation of speci men collections with clinical data about the individual from whom the specimen is derived; consent to specimen collection, transfer, storage, and use; authorization of use of protected health informa tion; pathologic data on the specimen (such as gross description and accompanying diagnosis)
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Snap-frozen (unfixed and Tonsil Microchip gel electrophoresis and immersed in RNA-stabilizing gene expression level via PCR buffer) , thawed for 0, 5, and 45 min, 1, 3, 6, and 16 h Snap-frozen, formalin-fixed RNA quality paraffin-embedded (FFPE)
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From page 45... ...
; RNA retrieved from pancreatic cancer samples with RIN of C7 subject to LCM yielded RNA suitable for further downstream applications ·Fresh-frozen pancreas tissue banked according to a standardized research protocol yields high-quality RNA in about 50 percent of specimens and can be used for enrichment with LCM; quality of tissues in the biobank was not adversely affected by slight variations in warm-ischemia times or different storage periods ·Comparison between RNA quality (RIN) and gene expression analysis Strand et al., shows dense clustering of high-quality samples but weak clustering of 2007 low-quality samples ·Manual and RIN methods are superior to ratio method ·RNA stable in both tissues under all conditions for up to 616 h Micke et al., ·Expression levels essentially stable when samples kept on ice 2006 ·Marked regulation of single genes observed during room-temperature storage in normal saline and RNA-stabilizing buffer ·RNA from 54 of 47 samples had proper ribosomal peaks ·Nonfixed specimens may be transported on ice for hours with minimal influence ·Minimal RNA degradation after 30 min Botling et al., ·Relevant changes in some gene-expression levels at 45 min 2009 ·Repetitive thawing cycles had similar effects on RNA integrity ·Incubation in RNA-stabilizing buffer prevents RNA degradation ·Introduced heating into extraction protocol to improve quality; Li et al., 2007 incubation at 70°C for 20 min was applied to disrupt cross-links in FFPE without compromising RNA integrity ·TaqMan detection influenced by master mix, amplicon size, and use of preamplification step ·Comparable results in frozen and FFPE tissue ·Provided PCR protocol for gene-expression analysis Votavová et ·62 of 65 samples "successfully" analyzed al., 2009 continued
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46 FUTURE USES OF THE DOD JPC BIOREPOSITORY TABLE 2-1 Continued Collection or Preservation Methods Tissue Types Attributes FFPE Parathyroid Proteome quality Fresh-frozen, FFPE Colon adenoma Proteome quality Liquid chromatography Frozen, FFPE Frozen/optimal Proteome quality cutting temperature Liquid chromatography (OCT) -embedded livers (rats)
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From page 47... ...
Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization." ·"Archival samples displayed a modest increase in methionine oxidation, from approximately 17 percent after one year of storage to approximately 25 percent after 10 years." ·"These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery." ·"Comparable molecular mass representation was found in extracts Scicchitano et from FFPE and OCT-frozen tissue sections, whereas protein yields al., 2009 were slightly less for the FFPE sample." ·"The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM [laser capture microdissection] did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples." ·"Subcellular representation in FFPE samples was similar to OCT frozen, with predominantly cytoplasmic proteins identified.
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Proteome quality FFPE Liver, kidney, heart, RNA quality (different temperatures brain, lung, spleen [4, 2025, 37°C] and storage (rat)
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From page 49... ...
·RNA quality not adversely affected by long interaction with fixative ·RTPCR quality is affected by long interaction with fixative ·Sample size influences quality: the thicker the sample, the longer it takes for fixative penetration and the lower the RNA quality; similarly for RTPCR ·Optimal fixation period 1224 h, yielded best RNA. Chung et al., 2008 improvements in reproducibility and dynamic range, but more traditional immunohistochemistry for protein detection still has a great role in diagnostic pathology.
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Data obtained with these techniques are likely to be incorporated into the clinical workflow as image analysis migrates from the microscope to the computer. Proteome Analysis The emergence of proteomic methods has enabled researchers to interrogate expressed proteins from a number of different tissue types systematically.
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Gene-Expression Profiling Since the method of gene-expression microarrays was developed in the mid-1990s, genomewide expression profiling has been used widely in research (Janssens and van Duijn, 2008; Kraft et al., 2009; van der Net et al., 2009; Xu et al., 2009)
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-have been designed to work with mRNA extracted from FFPE tissue (Beck et al., 2010; Wu et al., 2010)
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. The isolation of mRNA compared with miRNA can be complicated by substantial RNA degradation.
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Elemental and Chemical Studies Tissues are routinely analyzed for trace minerals in clinical pathology laboratories for diagnostic purposes. Trace minerals are preserved and can easily be localized and measured in FFPE tissues.
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Adequacy of fixation of a pathologic specimen is determined largely according to the dimensions of the tissue (when it is submerged in formalin in the gross state and when it is sampled for single aliquots to be individually processed into tissue blocks, formalin penetrates about 1 cm into tissue) and the total time in fixative.
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in the residual tissue in a block. The lack of quality control of pathologic tissue blocks made available for research, to verify the nature and content of the remaining tissue, can detract from their usefulness for research.
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Failure to reclassify cases correctly according to current diagnostic standards and current diagnostic terminology for any of the above reasons may skew research data. Preanalytic variations related to preoperative or intraoperative factors may create molecular artifacts.
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The committee does not know whether or how many specimens in the JPC repository are annotated with data related to perioperative variables that may be pertinent to molecular research on the molecular profiles of tissue samples. Perhaps the most common preanalytic variable with an important effect on results of analytic tests, such as immunohistochemical staining, is the time that elapses after surgical removal of tissue until it is stabilized with fixation or freezing (cold ischemia time)
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The lack of relevant clinical data elements limits the value of the molecular analysis data for prediction and the conclusions that can be drawn. The type and amount of clinical data provided by the physician requesting consultation was not prescribed in a standardized fashion by the JPC repository.
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2009. Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue.
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2008. Tissue handling and speci men preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue.
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18 months experience of peer review-based quality assessment in an English teaching hospital. American Journal of Surgical Pathology 16(5)
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2007. RNA quality in frozen breast cancer samples and the influence on gene expression analysis -- a comparison of three evaluation methods using microcapillary electrophoresis traces.
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The American Journal of Surgical Pathology 24(7)
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