Tissue preparation for immunocytochemistry.

Author(s): Williams JH, Mepham BL, Wright DH

Publication: J Clin Pathol, 1997, Vol. 50, Page 422-8

PubMed ID: 9215127 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated potential effects of a pre-fixation delay (0-8 h), as well as fixative type, pH, volume, and fixation temperature and duration on the immunohistochemical staining of 5 analytes in tonsil.

   Summary of Findings:

   Superior antigen preservation and staining consistency were observed in specimens preserved with 10% formal saline, 10% neutral buffered formalin, and 10% zinc formalin, while inferior results were observed among specimens preserved with 10% formal acetic, B5, and Bouin's fixative. For specimens preserved with 10% neutral buffered formalin, fixation for 12-36 h at ambient temperature, with a solution pH of 5-7 produced optimal immunostaining for the majority of antigens investigated. Increasing either the temperature or duration of fixation produced similar antigen-specific results. A pre-fixation delay, fixative to tissue ratio and a methanol additive (0-5%) did not significantly alter immunostaining in formalin fixed specimens.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Formalin
   • Other Preservative

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Time in fixative	<5 h 5 h 12 h 24 h 36 h 48 h
   Biospecimen Preservation	Temperature of fixation/preservation	18 degrees C 27 degrees C 37 degrees C
   Biospecimen Preservation	Fixative pH	11 3 5 7 9
   Biospecimen Preservation	Tissue to fixative ratio	1:1 1:20
   Storage	Time at room temperature	0 h 1 h 2 h 4 h 8 h
   Immunohistochemistry Specific	Targeted peptide/protein	CD20 CD45RO CD3 Vimentin Kappa
   Biospecimen Preservation	Type of fixation/preservation	B-5 fixative Bouin's fixative Carson's fixative Formalin (buffered) Formalin (unbuffered) Formol acetic acid Formol saline Zinc formalin
   Biospecimen Preservation	Fixative additive/buffer	Methanol None Multiple concentrations evaluated

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2. Study Purpose

   This study investigated the effects of variables associated with tissue dehydration and paraffin embedding, including the type of clearing reagent, embedding media, and automated tissue processor used, and the duration and temperature of processing, on the immunohistochemical staining of 5 analytes in tonsil.

   Summary of Findings:

   Optimal immunostaining intensity was observed for the majority of antigens when longer dehydration and paraffin impregnation durations (10 and 8 h, respectively), and an elevated processing temperature (45 degrees C compared to ambient temperature) were employed. Other parameters investigated (the type of tissue processor and reagents, the duration of clearing) did not significantly affect the quality of immunostaining.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Embedding medium	Ames Tissue Tek II Ames Tissue Tek III Fibrowax Paramat extra Lambwax
   Biospecimen Preservation	Embedding duration/condition	2 h 4 h 8 h
   Immunohistochemistry Specific	Targeted peptide/protein	CD20 CD45RO CD3 Vimentin Kappa
   Biospecimen Preservation	Dehydration duration/condition	2 h 7.5 h 10 h 45 degrees C Ambient temperature
   Biospecimen Preservation	Clearing duration/condition	45 degrees C Ambient temperature
   Biospecimen Preservation	Clearing agent	Chloroform Clearene Xylene Xylene substitute

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3. Study Purpose

   The purpose of this study was to determine the influence of the temperature and duration of tissue section drying and storage on the immunoreactivity of 11 analytes. Formalin-fixed paraffin-embedded tonsil, colon, uterus, and breast specimens were analyzed.

   Summary of Findings:

   Immunostaining intensity was adversely affected in tissue sections dried longer than 8 h at 60 degrees C or at 70 degrees C for as little as 30 minutes. Optimal immunostaining was observed in tissue sections dried overnight at 37 degrees C. Storage of FFPE sections on slides for up to 6 months at 4 degrees C or ambient temperature did not affect immunoreactivity, with the exception of desmin.

   Biospecimens

   • Tissue - Tonsil
   • Tissue - Uterus
   • Tissue - Breast
   • Tissue - Colorectal

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Tissue section adhesion	37 degrees C 60 degrees C 70 degrees C 30 min 1 h 2 h 4 h 8 h
   Storage	Storage temperature	Ambient temperature 4 degrees C
   Storage	Storage duration	1 d 7 d 1 month 3 months 6 months
   Immunohistochemistry Specific	Targeted peptide/protein	CD20 CD45RO CD3 Vimentin Kappa MIB1 PC10 1D5 BCL-2 alpha-SMA CEA

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