Previously Stained Slides for Use with Immunohistochemistry: A Single Institution Study for Consult Cases and Small Biopsies
Mathew D Rumery, David J Davis, Scott Lucia. University of Colorado, Aurora, CO
Background: Limited tissue is often available for the use of immunohistochemistry (IHC) in the case of small biopsies, where the block may be easily exhausted. In such instances, special stains may have been prepared that are of limited value relative to a desired IHC marker. While de-staining of prior stained slides can be used in these cases, the optimal stains for use and the prior stain's effect on signal intensity have not been previously reported. Here we report a semi-quantitative analysis of IHC intensity when performed on previously stained and de-stained slides.
Design: Using archival tissue, we stained slides with commonly used special stains for a number of disciplines often affected by limited tissue including neuropathology, hepatopathology, hematopathology, breast, and cytopathology. The stains used included negative IHC controls, hematoxylin and eosin (H&E), iron, trichrome, acid fast bacilli, periodic acid Schiff (PAS), PAS with diastase (PAS/D), Congo Red, and PAS Orange G. These previously stained slides then had the coverslip removed and were de-stained using routine methods. Subsequent to de-staining, the following IHC was performed: AE-1/AE-3, CK 7, CK 20, IDH-1, Mib-1, synaptophysin, CD 3, CD 20, CD 34, bcl-2, bcl-6, WT-1, BerEp4, ER, and PR. For each stain, a 'gold standard' control was prepared using our institution's procedure for IHC. The intensity of IHC signal for each de-stained slide was then compared to this 'gold standard' and given a semi-quantitative score (0-3+).
Results: Nine special stains and 15 antibodies were evaluated in total. Significant variation in IHC signal intensity was observed between both stains and specific antibodies. Despite the variation, some stains persistently resulted in stronger IHC signal intensity. Slides de-stained from trichrome, negative IHC controls, and H&E resulted in the strongest staining intensity on average. PAS/D resulted in the least intense signal, and the staining that was present showed a loss of specificity.
Conclusions: De-staining trichrome, negative IHC control, and H&E stains with subsequent IHC resulted in the strongest signal with varying results of other stains tested. We hypothesize that the amount of chemical alteration of the protein moieties caused by the stain impacts the staining intensity, as demonstrated by the poor intensity and specificity of the IHC on PAS and PAS/D. Details of staining and de-staining procedures will be discussed.
Category: Quality Assurance
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 245, Tuesday Morning