Quantitative immunohistochemistry reproducibility

Immunohistochemistry (IHC) has advanced considerably since the first edition of this handbook was published in 1983 (1), and the driving force behind that change has been the need for standardization. If tissue staining is to provide consistent, reproducible diagnostic information, it must continue to evolve from an art form to a science. That evolution demands quantitation and reproducibility of methodology, and extending from that, consistency of results. 

Figure 5.3 Diagram depicts the further-designed studies to test our hypothesis with respect to standardization of immunohistochemistry based on the antigen retrieval technique exemplified in a multiple direction to draw a conclusion, (a) Periods of formalin fixation, (b) Variable delay of fixation, (c) Storage of FFPE tissue blocks or sections, (d) Variable thickness of FFPE tissue sections, (e) Other variable conditions of processing FFPE tissue blocks. The stereoscopic frame of a cube represents the reliable limitation of quantitative IFIC demonstrated by serial studies as recommended in the text. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2007 55 105-109.

It may be helpful to consider the following criteria when evaluating a proposed positive control for immunohistochemistry. A positive control is ideally (1) reproducible and quantitative, (2) available in unlimited quantities, (3) antigen-specific, (4) inexpensive, and thereby adaptable for mass production, and (5) stable over time. Judged against these criteria, current practice falls short of the ideal. Biopsy materials are variable, both from patient to patient and even within a single tumor sample. Biopsy or resection materials are also obviously not available in unlimited quantities. The need for (microtome) sectioning of each positive control also creates a labor cost that is often under-appreciated by many clinical IHC laboratories. 

Presently, immunohistochemistry requires improvements in quality, reproducibility, speed, quantitation, and standardization. Some of these goals can be achieved by using computerized bar code-driven automatic immunostainers that automatically dispense reagents, control washing, mixing, and heating to optimize immunohistochemical reaction... 

Note that although immunohistochemistry is contributing significantly to clinical information, in the absence of quantitation, this method is subjective and prone to intra- and interobserver variations. To assure the reproducibility of histopathological results and then-correct evaluation, at least an assessment of the percentage of positive cells and both the staining pattern and intensity must be made. If available, computer-assisted image analysis... 

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