Tissue identity testing

In pathology practice, DNA-based tissue identity testing is typically used to detect tissue contaminants or floaters and for mislabeled specimens. It is particularly... 

The PCR-based tissue identity test requires a very small amount of DNA and can be successfully performed using FFPE tissue samples or hematoxylin and eosin-stained tissue fragments removed from glass slides. 

Samples for identity testing can be any specimen that contains DNA. Samples obtained from an individual for paternity testing or as a reference sample to be compared with DNA prepared from evidence are usually peripheral blood or buccal mucosa. Samples useful for forensic testing, engraftment assays, and the identification of clinical samples may range from plucked hairs to bone marrow aspirates to paraffin embedded tissue. While subject to degradation over time in the presence of enzymes, acidic or basic conditions, or high temperature, DNA is a remarkably stable molecule that can be recovered and successfully analyzed from solutions, surfaces, and cells. 

Figure 3.3 Comparison of array CGH among DNA extracted from fresh tissue, FFPE tissue by heating protocol or nonheating protocol for two human tissue samples of metastatic carcinoma in lymph node (a-c), and undifferentiated non-small cell carcinoma (d-f). Array CGH hybridization genomic profiles show ratio values representing relative copy number of single BACs. A good result is scored as 1.0 that indicates a low standard deviation for gains (>0.2), normal (0.0), or losses (<-0.2). In these two cases, fresh samples show best score as 2, both FFPE tissue samples show identical score of 3. Each spot represents the average of three replicates. Clones are ordered by chromosomal position as numbers at the bottom (x axis) of each picture. The y axis is the log2 ratio of test reference intensity. Provided by Sandy DeVries from Dr. Frederic Waldman s Lab at UCSF.

Two different and possibly complementary approaches have been explored. One utilizes a panel of quantifiable internal reference standards (QIRS), which are common proteins present widely in tissues in relatively consistent amounts.11,22 In this instance because the reference proteins are intrinsic to the tissue they are necessarily subjected to identical fixation and processing, and incur no additional handling or cost, other than synchronous performance of a second IHC assay (stain), such that the intensity of reaction for the QIRS and the test analyte can be compared by IA, allowing calculation of the amount of test analyte (protein) present on a formulaic standard curve basis. The other approach seeks to identify external reference materials and to introduce these into each step of tissue preparation for cases where IHC studies are anticipated in this instance the logistical issues of production, distribution, and inclusion of the reference standard into all phases of tissue processing also must be considered, along with attendant costs. 

B[/ ]P metabolites have been shown to bind to DNA in culmred human hepatocytes and in human bladder and tracheobronchial explants. The metabolites identified were identical to those produced in other species and differed only in the relative percentages of formation. Human tissues were most active in metabolizing P>[a P and exhibited at least a threefold higher covalent binding of metabolites to DNA than hamsters, dogs, monkeys, or rats. In addition, P>[a P has been tested extensively in several bacterial and mammalian cell systems and has been chosen as a positive control for the validation of some of these systems. ... 

The FDA investigators have the authority to collect samples as described under the comphance program 7348.808. Samples of a test article, the carrier, the control article or test and control article mixtures may be selected and sent to FDA laboratories to determine the identity, strength, potency, purity, composition, or other characteristics that wiU accurately define the coUected sample. In fact, even physical samples such as wet tissues, tissue blocks, and slides may be collected. When the field investigator collects a sample of any chemical substance, he will also coUect a copy of the methodology from the sponsor of the testing facihty. The copy of the methodology will be sent to the FDA laboratory selected to perform the sample analysis. 

The most appropriate control for any immunostain would be an internal control because it would have been subjected to identical pre-analytical and analytical variables as the test tissue. However, such controls are invariably non-lesional or benign cells that express the antigen of interest at levels different to the tumor cells and are thus not ideal controls. Nonetheless, they are currently the best controls available. 

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