Tissue FFPE

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.

If a monoclonal antibody was generated by immunization with a full-length native protein rather than a peptide, then the immunized mouse will generate antibodies that recognize both linear and conformationally dependent epitopes. Only a small subset of these monoclonal antibodies will likely be useful for clinical use on formalin-fixed, paraffin-embedded tissue (FFPE) samples. Those that are useful tend to have epitopes that are linear the epitopes are not dependent on the protein s three-dimensional conformation (see Chapter 16). Therefore, for antibodies generated in response to immunization with full-length proteins, the peptides that serve as positive controls will be linear stretches of amino acids derived from the native protein sequence, as listed in protein databases. 

Reference FFPE FPPE Tissue Fresh Tissue FFPE Tissue ... 

THE PROMISE AND CHALLENGE OF SHOTGUN PROTEOMICS IN ARCHIVAL, FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUES (FFPE)... 

Archived tumor samples are available as either frozen or formalin-fixed, paralfin-embedded (FFPE) tissue. FFPE samples are usually more readily available than are frozen samples. 

The increasing attention directed to the adverse effects of variation in sample preparation upon the quality of IHC staining of FFPE tissues has served to reinforce the importance of determining the optimal AR method for each antibody/detection system/antigen to achieve optimal retrieval and optimal staining of tissues that may have been processed and stored in different and unknown ways (see Chapter 5 for details). Practically, in considering... 

Ferrier FFPE tissues of To vahdate AR-IHC A pretest based... 

Naito et al.18 FFPE tissues of To establish optimal Heating at two or... 

Choi et al.20 FFPE tissues of invasive aspergillosis from 16 pediatric cases, fixed in formalin for 6-72 h To establish an optimal AR protocol for mAb WF-AF-1 (Dako), testing three different retrieval solutions of pH 6.0, 8.0, and 10.0 with MW heating for 10 min Satisfactory IHC results are achieved using AR with high pH. 

Gill et al.21 Archival FFPE spinal cord tissue both paraformaldehyde-fixed frozen rat spinal cord tissue and paraffin-embedded same tissue To establish an optimal protocol for detection of low-abundance protein (NeuN) in human spinal cord FFPE tissue sections, testing three AR solutions of pH 6, alkaline, and acidic buffer, with three heating conditions 95,100, and 105°C Heating FFPE tissue sections in an alkaline buffer yields most effective AR-IHC staining results. 

TABLE 1.4 Comparison of pRB-IHC in 27 Cases of FFPE Tissues of Bladder Cancer Using Four Protocols of AR... 

Although the novel AR protocol using citraconic anhydride improved the intensity of IHC on FFPE tissue sections for more than half of the antibodies tested, compared to that achieved by other conventional AR protocols, not all antibodies benefitted, which would argue that the citraconic anhydride method does not serve as a truly universal AR protocol. Indeed, many investigators (Table 1.2) have concluded that different antigens may require different specific AR protocols. In this respect, the test battery is a convenient and cost-effective method for assessing the appropriate AR protocol.2,8 Nevertheless, the present data certainly support inclusion of the citraconic anhydride AR method in such a test battery. With respect to the two heating temperatures for citraconic anhydride, the ultimate choice of method for any laboratory may depend on the equipment available. 

In a study involving decalcified FFPE rat joint tissue sections and a variety of AR methods, Wilson et al.32 reported successful application of 0.2 M boric acid at pH 7.0 as the AR solution combining a low-temperature incubation (60°C for 17 h). The principal advantage of this AR protocol was that it minimized lifting or loss of decalcified hard tissue sections from charged slides. Their basic approach for establishing an optimal AR protocol was a test battery as described above. In a separate series of studies, based upon prior... 

Figure 1.2 Examples of immunostaining intensity from comparison of pRB-IHC in 27 cases of FFPE tissues of bladder cancer (Table 1.4). (A-D) Negative (<10%) showing a few weak positive nuclei (arrows) (E-H) moderate positive (>10%) (I-P) strong positive (>50%). Arrows indicate positive nuclear staining for some lymphocytes or other stromal cells as an internal control. Note the lack of nuclear hematoxylin counterstaining due to low pH AR treatment. The order of cases are indicated in Table 1.4. Reproduced with permission from Shi et al.. Biotech. Histochem. 2007 82 301-309. See color insert.

Recently, the use of AR has extended into several other areas, yielding interesting information for cytology, fresh cell/tissue sections, and fluorescence IHC (fluorescence in situ hybridization [FISH]), in addition to adaptations of the method for extraction of nucleic acids and proteins from FFPE tissues for use with modern methods of molecular analysis. In this chapter, the emphasis is on expanded applications in diagnostic cytology, fresh frozen cell/... 

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