Antigen retrieval techniques in immunohistochemistry: comparison of different methods.

Author(s): Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, Ascani S, Santini D, Piccaluga PP, Leone O, Damiani S, Ercolessi C, Sandri F, Pieri F, Leoncini L, Falini B

Publication: J Pathol, 1997, Vol. 183, Page 116-23

PubMed ID: 9370957 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of AR technique, type of fixative, duration of fixation, and IHC detection method on IHC staining in a variety of normal and pathological tissues including acute non-lymphoid leukaemia and intermediate cell neuroendocrine carcinoma. A panel of 61 antibodies was used for IHC staining of tissue.

   Summary of Findings:

   Detection of IHC staining using automated or manual APAAP methods was comparable to detection using a modified streptavidin-biotin complex immunoperoxidase method. Although antigen specific effects were noted, in general, HIAR yielded immunostaining results that were superior to the use of protease XIV, including less background staining, staining of otherwise undetectable antigens, and allowing for the use of a higher antibody working dilution. HIAR was equally as effective in formalin- and B5-fixed specimens and attenuated differences in staining seen between specimens fixed in formalin for 24 h and 1 week. After AR by protease XIV, IHC staining was particularly unpredictable in B5-fixed specimens compared to formalin-fixed specimens where 5 antibodies benefitted from proteolytic enzyme treatment. Generally speaking, for antigens where AR was not necessary to obtain adequate immunostaining, a lower working dilution of antibody was necessary compared to the working dilution used after AR. HIAR yielded superior IHC results when EDTA buffer was used compared to Tris-HCl and citrate buffer, the latter faring the worst. Notably, EDTA buffer was superior regardless of fixative (formalin versus B5), nature of the antibodies (monoclonal versus polyclonal), or source of heat (microwave versus pressure cooker). While no significant differences were noted between HIAR by microwave irradiation as opposed to pressure cooking, pressure cooking for 90 seconds was superior to 1 or 2 minutes.

   Biospecimens

   • Tissue - Bone Marrow
   • Tissue - Placenta
   • Tissue - Lymph Node
   • Tissue - Breast
   • Tissue - Colorectal
   • Tissue - Stomach
   • Tissue - Prostate
   • Tissue - Endometrium
   • Tissue - Thyroid Gland
   • Tissue - Larynx
   • Tissue - Eye
   • Tissue - Muscle (Skeletal)
   • Tissue - Other
   • Tissue - Brain

   Preservative Types

   • Formalin
   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma
   • Neoplastic - Lymphoma
   • Neoplastic - Sarcoma
   • Neoplastic - Leukemia

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	B-5 fixative Formalin (buffered)
   Biospecimen Aliquots and Components	Type of slide	Silane-coated Naturally charged
   Analyte Extraction and Purification	Antigen retrieval	None Protease digestion HIAR in citrate buffer HIAR in Tris-HCl buffer HIAR in EDTA-NaOH
   Analyte Extraction and Purification	Temperature of heat-induced retrieval	Pressure cooker 1 min Pressure cooker 90 sec Pressure cooker 2 min Microwave
   Immunohistochemistry Specific	Detection method	Manual APAAP Automated APAAP Modified streptavidin-biotin complex immunoperoxidase
   Biospecimen Preservation	Time in fixative	2 h-2 h 30 min (B5) 24 h-1 week (formalin)
   Immunohistochemistry Specific	Antibody dilution/concentration	A broad range was investigated

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