Effects of fixation and processing on immunohistochemical demonstration of immunoglobulin in paraffin sections of tonsil and bone marrow.

Author(s): Curran RC, Gregory J

Publication: J Clin Pathol, 1980, Vol. 33, Page 1047-57

PubMed ID: 7002958 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 fixative and processing methods on PAP immunohistochemistry using tonsil and bone marrow tissue.

   Summary of Findings:

   Fixation of tonsil tissue using either phosphate-buffered formaldehyde or formol saline and treatment with trypsin resulted in good preservation and definition of histological structures. PAP immunostaining was clearer in thinner sections (2 to 4 uM) and in specimens embedded in paraffin rather than paraplast. Unsatisfactory results were obtained with sections thicker than 8 uM. Untrypsinized sections showed weak or no staining after fixation in phosphate-buffered formaldehyde or formol saline which was not improved by (a) leaving these tissues for up to 7 days in water, xylol, or chloroform, (b) increasing dehydration, clearing, or embedding duration by 3 days, or (c) post-fixation or prefixation incubation in phosphate buffer. However, several other fixatives did not require treatment with trypsin. Fixation using unbuffered formaldehyde resulted in weak PAP immunostaining that was further weakened by the addition of NaCl. Fixation with acetic acid-formol saline resulted in well preserved histological structure and good immunostaining, particularly with 2-10% acetic acid and fixation times less than 48 h. Fixation in mercuric chloride solution showed similar results to those seen with formalin and formol saline although cell shrinkage was also seen. Fixation in formol sublimate or Bouins fluid gave excellent preservation of histological structures and good PAP immunostaining particularly when fixation was carried out for 48 h. Fixation in Carnoys solution without chloroform resulted in slightly better immunostaining than with chloroform, however preservation of histological structures was the worst of all fixatives, although still adequate. The best immunostaining of bone marrow tissue was seen with a combination of prefixation in formol sublimate followed by fixation in 10% acetic acid-formol saline.

   Biospecimens

   • Tissue - Tonsil
   • Tissue - Bone Marrow

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Morphology	H-and-E microscopy
   Protein	Immunohistochemistry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Time in fixative	1 h 2 h 6 h 24-48 h 72 h 96 h
   Analyte Extraction and Purification	Protein digestion	Trypsin None
   Biospecimen Acquisition	Biospecimen location	Tonsil Bone Marrow
   Biospecimen Preservation	Embedding medium	Paraffin Paraplast
   Biospecimen Preservation	Clearing duration/condition	6-9 h 3 d Xylol Chloroform
   Biospecimen Preservation	Dehydration duration/condition	1 d 3 d
   Biospecimen Preservation	Embedding duration/condition	12-24 h 84-96 h
   Biospecimen Aliquots and Components	Type of slide	2 to 18 uM thick sections
   Analyte Extraction and Purification	Incubation duration/condition	Up to 7 days in water Up to 7 days in xylol Up to 7 days in chloroform
   Biospecimen Preservation	Type of fixation/preservation	Bouin's fixative Carnoy's solution Formaldehyde Formol saline Formol sublimate Mercuric chloride solution
   Biospecimen Preservation	Concentration of fixative	0.1% 0.5% 1% 2% 4%
   Biospecimen Preservation	Fixative additive/buffer	Acetic acid Walpole's buffer Chloroform Phosphate buffered saline (PBS) Sodium chloride Sucrose

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