Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques.

Author(s): Taylor CR, Shi SR, Chaiwun B, Young L, Imam SA, Cote RJ

Publication: Hum Pathol, 1994, Vol. 25, Page 263

PubMed ID: 7512074 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to compare different antigen retrieval solutions and microwave heating times among formalin-fixed, paraffin-embedded (FFPE) tissue sections fixed for different durations (12 h - 7 d) for the following nuclear antigens, estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), p53 protein, proliferating cell nuclear antigen (PCNA), and Ki-67 antigen.

Conclusion of Paper

The authors report obtaining the most consistent, strong immunostaining with low background by microwave heating sections for 10 minutes in 0.05 mol/L glycine HCl (pH 3.5) or in citrate buffer solution (pH 6) for a variety of tissues. Urea solution, distilled water, and lead thiocyanate solution were less universally effective. However, they report that longer fixation times (up to 7 days) generally required more vigorous retrieval protocols.

Studies

Study Purpose

The purpose of this study was to evaluate the effectiveness of different antigen retrieval solutions on restoring antigenicity in FFPE sections of estrogen receptor, progesterone receptor, androgen receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen, all of which are intranuclear antigens. These were examined in 40 different malignant neoplasms, including breast, colon, stomach, thyroid, lung, and prostate carcinoma, as well as lymphoma, melanoma, seminoma, and some sarcomas, as well as non-neoplastic tonsil.

Summary of Findings:

Antigen retrieval was necessary for all antigens evaluated. Optimal antigen retrieval methods were antigen specific.The authors report that sections pretreated with (a) lead thiocyanate displayed nuclear staining of variable intensity; (b) sodium citrate buffer resulted in the least variable nuclear staining with greatest contrast to background; (c) urea yielded acceptable results with all antigens but considerable background was present in some tissues; (d) distilled water resulted in moderate to poor staining, (e) glycine HCL buffer (without detergent) resulted in non variable nuclear staining with high contrast to background, although the addition of detergent compromised morphology. No differences were observed between antibody visualization with the supersensitive biotin streptavidin or avidin-biotin conjugate kits. Microwave heating for 10 minutes was sufficient in all cases. Optimal results were obtained with tissue fixed for 12 - 24 hours.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma
Neoplastic - Lymphoma
Not specified
Neoplastic - Melanoma
Neoplastic - Other
Neoplastic - Sarcoma

Platform:

Analyte	Technology Platform
Protein	Immunohistochemistry

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Antigen retrieval	Glycine HCl buffer Lead thiocyanate Sodium citrate buffer Urea Distilled water Glycine HCl buffer with detergent
Biospecimen Preservation	Time in fixative	12 h 24 h 3 d 7 d
Analyte Extraction and Purification	Temperature of heat-induced retrieval	3 min of microwave heating 5 min of microwave heating 10 min of microwave heating 20 min of microwave heating
Immunohistochemistry Specific	Detection method	Supersensitive biotin streptavidin kit Avidin-biotin conjugate kit

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