The effect of different fixatives and length of fixation time on subsequent AgNOR staining for frozen and paraffin-embedded tissue sections.

Author(s): Rowlands DC, Ayres JG, Crocker J

Publication: Histochem J, 1993, Vol. 25, Page 123-32

PubMed ID: 7682206 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 preservation method and duration on AgNOR staining appearance in tonsil specimens. Specimens were snap-frozen, snap-frozen and fixed, fixed and processed, or processed without fixation prior to staining. Snap-frozen specimens were stored in liquid nitrogen for 1-14 days. Fixed frozen sections were stored at room temperature in a desiccator for 1-7 days prior to staining. Paraffin blocks were stored at room temperature for 1-8 weeks prior to sectioning, and paraffin sections were stored overnight at room temperature prior to staining.

   Summary of Findings:

   Adequate AgNOR staining was seen in all tissue samples, regardless of preservation method, or time in fixative. Statistical analysis was not performed due to small sample size (only 6 specimens were preserved using any one protocol). Greater non-specific AgNOR staining and poorer cytoplasmic morphology was seen in unfixed frozen sections and in paraffin sections that had not been previously fixed. In general, fixed frozen specimens showed clearer AgNOR staining and less precipitate than fixed paraffin-embedded specimens. Acetone and alcohol fixatives resulted in less non-specific nuclear chromatin staining in frozen sections than in paraffin-embedded ones. However, aldehyde-fixed (gluteraldehyde, formol-saline and formalin) specimens, which had generally weaker AgNOR staining than acetone or alcohol-based ones, showed more non-specific nuclear chromatin staining in frozen sections than in paraffin-embedded ones. Longer fixation times tended to result in increased precipitate, more pronounced stromal staining, and decreased resolution along with weaker staining of the AgNORs in aldehyde-fixed specimens, but had little effect on non-specific nuclear chromatin staining.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Frozen
   • Other Preservative
   • Ethanol

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Morphology	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Acetone Bouin's fixative Carnoy's solution Ethanol Formalin (buffered) Formol saline Glutaraldehyde Methanol Snap frozen
   Biospecimen Preservation	Time in fixative	15 min 30 min 60 min 3 h 6 h 24 h 48 h 72 h 96 h

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2. Study Purpose

   The purpose of this study was to determine the effects of delayed fixation in formol saline and the use of different tissue processors on AgNOR staining in tonsil specimens. Paraffin blocks were stored at room temperature for 1-8 weeks prior to sectioning, and paraffin sections were stored overnight at room temperature prior to staining.

   Summary of Findings:

   When fixation in formol saline was delayed for more than 2 d, there were large decreases in the resolution and darkness of AgNOR staining and a reduction in morphological preservation. Gradual increases in precipitate and non-specific nuclear staining were also observed as cold ischemia time increased from 15 min to 4 d. The Shandon 2LE processor (10 h dehydration, 3 h clearing, 5 h paraffin impregnation) yielded superior AgNOR staining results compared to the Miles Scientific VIP 2000 processor (6 h dehydration, 1.5 h clearing, 3 h paraffin impregnation).

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Morphology	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Dehydration duration/condition	6 h, 40 degrees C, intermittent vacuum 10 h, room temperature, no vacuum
   Biospecimen Preservation	Clearing duration/condition	1.5 h, 40 degrees C, intermittent vacuum 3 h, room temperature, no vacuum
   Biospecimen Preservation	Embedding duration/condition	3 h, 60 degrees C, intermittent vacuum 5 h, 60 degrees C, under vacuum
   Biospecimen Preservation	Duration of tissue/ specimen processing	10.5 h (Miles Scientific VIP 2000 processor) 18 h (Shandon 2LE processor)
   Biospecimen Acquisition	Cold ischemia time	15 min 24 h 48 h 72 h 96 h

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