Inadequate formalin fixation decreases reliability of p27^Kip1 immunohistochemical staining: Probing optimal fixation time using high-density tissue microarrays

Immunohistochemical analysis of molecular targets in clinical tissues is increasingly becoming central to our ability to render diagnoses, to predict prognosis, to select patients for appropriate therapies, and to provide surrogate end points for therapeutic monitoring. For example, reduction of immunohistochemical staining for the cyclin-dependent kinase inhibitor p27^Kip1 has been proposed as a potential prognostic biomarker in prostate, breast, and gastrointestinal tumors. We observed that with our standard formalin fixation in rapidly processed (same-day) radical prostatectomy specimens, there is often a gradient of p27^Kip1 staining in normal prostate epithelium, with more staining near the periphery and less staining toward the center of the sample. This raised the hypothesis that the reliability of staining for p27^Kip1 is decreased in inadequately fixed tissues. The implications of this, if true, are that many studies using p27^Kip1 for prognostic purposes may be subject to unpredictable artifacts, and hence unreliable results, if the fixation of the specimens is not well controlled. The objectives of the present study were (1) to formally test the hypothesis that inadequate fixation time is responsible for apparent loss of p27^Kip1 nuclear staining and (2) to test a recently proposed method for improving the uniformity of immmunohistochemical staining using formalin injection. Prostate tissue sections from radical prostatectomy specimens were either processed immediately (zero time fixation) or fixed for 1, 2, 3, or 8 days in 10% neutral buffered formalin before processing into paraffin. To assure identical antigen retrieval and immunohistochemical staining conditions for specimens fixed for different lengths of time, 2 high-density tissue microarrays (TMAs), containing 564 tissue samples (0.6 mm in diameter) were constructed. Based on an estimate of the percentage of nuclei in normal prostatic epithelial secretory cells with strong staining, quality of p27^Kip1 staining was graded in a blinded fashion with respect to fixation time. There was a significant increase in the percentage of cores that were scored as "strong" as fixation time increased from 0 (same-day processing) to 1 or more days (P < .0001). Interestingly, even at 8 days of fixation, there was excellent staining that was superior to the same-day processing. Based on these results, we conclude the following: (1) for large clinical specimens that have been fixed briefly to decrease diagnostic turn-around time, the reliability of interpretation of immunohistochemical staining may be quite limited; (2) for p27^Kip1, decreased antigen staining as a result of the widely held concept of "overfixation" is much less of a problem than "underfixation"; (3) formalin injection produces a marked improvement in staining for several markers, including p27^Kip1; and (4) high-density TMAs, which assure identical test conditions, provide an excellent platform on which to evaluate the effects of tissue fixation on immunohistochemical staining.