Elsevier

Human Pathology

Volume 33, Issue 7, July 2002, Pages 756-760
Human Pathology

Original Contributions
Inadequate formalin fixation decreases reliability of p27Kip1 immunohistochemical staining: Probing optimal fixation time using high-density tissue microarrays*,**

https://doi.org/10.1053/hupa.2002.126187Get rights and content

Abstract

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 p27Kip1 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 p27Kip1 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 p27Kip1 is decreased in inadequately fixed tissues. The implications of this, if true, are that many studies using p27Kip1 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 p27Kip1 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 p27Kip1 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 p27Kip1, 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 p27Kip1; 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. HUM PATHOL 33:756-760. Copyright 2002, Elsevier Science (USA). All rights reserved.

Section snippets

Specimen handling and TMA construction

In our standard approach to radical prostatectomy sectioning, the gland is inked, and the specimens are fixed for 1 to 4 hours in formalin by simply placing the specimen in a minimum of 15 volumes of 10% neutral buffered formalin. Prostate specimens are then sectioned from apex to base into 3- to 5-mm slices, and each slice is further sectioned into halfs or, more commonly quadrants. Tissues are submitted into standard-sized cassettes, and are placed onto our automated tissue processors the

Results

The gradation of staining seen with the standard sections from a radical prostatectomy sample sectioned and cut using our routine protocol of same-day tissue processing is illustrated in Fig 1.

. “Outside-in” gradation of staining for p27Kip1 in normal prostate epithelium. (A) Low-power view of staining near periphery of the specimen. Area marked “outside” is within 0.5 mm of the inked surgical margin. Note strong nuclear staining toward the outside of the gland and weak staining toward the

Discussion

In this study, we show that immunohistochemical staining against p27Kip1 protein using radical prostatectomy specimens in formalin-fixed tissue may mistakenly appear to be downregulated as a result of an artifact relating to inadequate tissue fixation. Contrary to popular dogma in the pathology field, prolonged fixation, even up to 8 days, produced no decrement in staining for p27Kip1. Although TMAs are not necessary to carry out studies on the effects of fixatives on immunostaining, they do

Acknowledgements

The authors thank Geert van Leenders for illustrating, first-hand, the method of formalin injection of radical prostatectomy specimens.

References (20)

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Supported by grants from Public Health Services NIH/NCI Specialized Program in Research Excellence (SPORE) in Prostate Cancer, Johns Hopkins (P50CA58236), and The University of Michigan (P50CA69568).

**

Address correspondence and reprint requests to Angelo M. De Marzo, MD, PhD, Johns Hopkins Medical Institutions, Department of Pathology, Division of Genitourinary Pathology, Bunting/Blaustein Cancer Research Building, Room 153, 1650 Orleans St, Baltimore, MD 21231-1000.

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