Fixation and Processing

Routine formol-calcium fixed, paraplast-embedded, five micron thick, tissue sections were generally used throughout our studies, but cryostat- or paraplast-embedded sections fixed in any manner that does not interfere with vicinal diols may be used equally well. Sections must be floated onto clean, grease-free slides. This should avoid the loss of sections during staining. Chrome alum gelatin (see below) may be used if sections become detached. 

Method for Preparing Gelatin—Chrome Alum Adhesive Chrome-Alum Gelatin 

Add 2g of gelatin to 30ml of cold water to soften gelatin then add 170ml of boiling water, mix and cool to room temperature. Add 0.2 g of chrome alum and shake gently to dissolve. 

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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. 

There are a number of different cell fixation and process methods used in laboratories worldwide. Fixation time in tissue will reflect a commonly accepted fixation time such as that seen in pre-analytical guidelines published in the package inserts for commercially available kits. Regarding cell lines points to note are how soon are the cells fixed after harvesting, are the cells fixed in suspension, or when are they in a suspension matrix such as agarose. 

The thicker the specimen, the longer it takes for penetration to be complete. A 1-mm thick piece of tissue will take a certain length of time to become penetrated. A 2-mm piece of the same tissue will take four times as long (double squared), and a 3-mm piece will take nine times as long (triple squared). For very thick gross specimens, complete penetration may not occur in any reasonable period of time. If you want very rapid fixation and processing, specimen thickness must be kept as thin as possible. 

The procedures described represent guidelines optimal methods should be determined in individual laboratories. This is because of the variability of tissue fixation and processing and the variety and stability of antigen targets. 

Improper fixation and processing Proper antigen fixation is the cornerstone of immunoperoxidase techniques, for without it, results will be poor. Some markers may be destroyed by certain fixatives overfixation may mask certain antigens (see Chapter 8) (4). Review manufacturer s package inserts for appropriate fixation techniques. Paraffin-embedded tissue should never be exposed to temperatures >60°C as this can destroy some antigens. 

Improper fixation and processing of unknown This emphasizes the need for controls and nnknowns to be processed in an identical manner. 

Werner, M., Chott, A., Fabiano, A., and Battifora, H. 2000. Effect of formalin tissue fixation and processing on immunohistochemistry. Am. J. Surg. Pathol. 24 1016-1019. 

Microtissue arrays are a possible solution to the limited supply of control tissue. Microarray blocks allow the incorporation of 200-300 fine tissue cores into blocks that can be used for controls against a wide variety of antibodies and as the cores are small (0.5-1.5 mm diameter) much of the original tissue block remains preserved. Microtissue arrays should be used with the recognition that each core of tissue has been subjected to different fixation and processing so that the level of antigen preservation in each of the 200-300 tissue samples are different and by no means standardized. 

One of the last of these IHC art forms is tissue fixation and processing. Laboratory professionals are little closer to uniformity in this part of the process, and achieving that uniformity, or standardization, remains one of the true unknowns in diagnostic interpretation. 

Studies using TEM and in situ precipitation to follow the pathway of topically applied compounds have focused on distinguishing between the intracellular and intercellular routes of transport of substances across the SC. In 1968, Silberberg [24] first used this technique to provide evidence that mercury, after topical application of 0.1% aqueous mercuric chloride, traverses across the SC in vitro via the intercellular spaces. But difficulties with fixation and processing prevented demonstration that the mercury aggregates were also present in the SC cells. Thus, the possibility that mercury may also have taken a transcellular route through the SC could not be excluded. 

The perceived need to identify objective markers to supplement, or conceivably supplant, the more subjective established histologic parameters has been a major driving force behind biomarker discovery efforts. It is crucial to recognize and account for the potential variability that can exist even with the new molecular parameters. Sources of variability include differences in molecular technique methodologies, tissue fixation and processing, interobserver and intraobserver variability (in immunohistochemistry-based biomarkers), and differences in cutoff points. Furthermore, illustration of statistical significance for a particular biomarker does... 

A new, very timely chapter on immunocytology has been included by Dr. Chivukula, which discusses proper cytologic technique for fixation and processing specimens obtained for hormone receptors and HER2/neu testing. 

There are several new completely rewritten chapters with new authors, all of them experts in their respective fields, including N. Volkan Adsay, Jonathan Epstein, Alyssa M. Krasinskas, Alvin W. Martin, George Netto, and Yuri E. Nikiforov. The latest recommendations for proper fixation and processing of hormone receptor testing are authoritatively discussed by Dr. Clive R. Taylor. 

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