Influence of decalcifying agents on immunoreactivity of formalin-fixed, paraffin-embedded tissue.

Author(s): Matthews JB, Mason GI

Publication: Histochem J, 1984, Vol. 16, Page 771-87

PubMed ID: 6206029 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 decalcifying agents and fixation with formalin-acid mixtures on FFPE specimens.

   Summary of Findings:

   Increasing decalcification time lead to increased susceptibility to trypsin digestion for all decalcifying agents, except Jenkin's fluid. Saline, EDTA, acetic acid, and D-califier (2) did not impair nuclear counterstaining while Von Ebner's fluid, Jenkin's fluid, nitric acid, and D-calcifier (1) did. Formic acid also showed some inhibition of nuclear counterstaining after 72 and 144 hour incubations. Immunostaining results were antigen specific and depended on decalcifying agent, incubation time, and duration of trypsin digestion. In general, the best staining, in terms of intensity and preservation of morphological structures, was seen when tissues were decalcified in neutral EDTA, formic, or acetic acid while worse staining was seen when hydrochloric acid containing decalcifying agents were used including Von Ebner's fluid, Jenkin's fluid and D-calcifier (1). Fixation with formic acid-formalin mixtures gave worse immunostaining results than those obtained when tissues were fixed in neutral buffered formalin and decalcified with formic acid after fixation. However, fixation with acetic acid-formalin mixtures gave strong immunostaining results without trypsinization.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Protein	Immunohistochemistry
   Morphology	Light microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Fixative additive/buffer	Acetic acid Saline Formic acid
   Analyte Extraction and Purification	Protein digestion	Trypsin 0 min 5 min 10 min 15 min 30 min
   Analyte Extraction and Purification	Incubation duration/condition	Decalcification 0 h 1 h 4 h 8 h 12 h 24 h 48 h 72 h 144 h 168 h 7 weeks
   Immunohistochemistry Specific	Targeted peptide/protein	IgG IgA IgM Lysozyme Factor VIII Keratin Lambda chains Kappa chains
   Analyte Extraction and Purification	Decalcification solution/ duration	NaCl EDTA Von Ebners fluid Jenkins fluid Formic acid Acetic acid Nitric acid D-calcifier (1) D-calcifier (2)

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