Effect of Decalcification and Fixation in Paraffin-Section Immunohistochemistry

Author(s): Arber Janet M, Arber Daniel A, Jenkins Kay A, Battifora Hector

Publication: Applied Immunohistochemistry, 1996, Vol. 4, Page 241

Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of duration of fixation and decalcification on IHC of multiple different FFPE tissue specimens. A panel of 57 antibodies was studied.

   Summary of Findings:

   53 out of 57 antibodies used for IHC were unaffected by any decalcification solutions or formalin fixation times of up to 72 hours. ER, PR, p53, Ki-67, and BerEp4 immunoreactivity was decreased or absent compared to non-decalcified control tissue when RBD or S/P Decal were used for decalcification, regardless of fixation time. Ki-67 and BerEp4 immunostaining were also weaker when Versenate was used for decalcification. The decreased immunostaining for Ki-67 was only after formalin fixation for 6 h. However, for BerEp4, fixation times of 6 and 24 h resulted in weaker immunostaining compared to controls, but 72 h-fixed specimens showed enhanced staining compared to non-decalcified controls. Changes in IHC staining intensity were variable based on tissue type used. Changes in antigen retrieval treatments for ER, PR, p53, and Ki-67 staining did not significantly alter outcomes. In general, H&E staining revealed preserved morphology after decalcification and up to 72 hours of formalin fixation, however, slight basophilic appearance, nuclear hyperchromasia, and loss of definition of cytoplasmic membranes and nuclear detail were noted.

   Biospecimens

   • Tissue - Bone
   • Tissue - Uterus
   • Tissue - Pancreas
   • Tissue - Spleen
   • Tissue - Colorectal
   • Tissue - Prostate
   • Tissue - Breast
   • Tissue - Lymph Node

   Preservative Types

   • Formalin

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma
   • Neoplastic - Lymphoma
   • Neoplastic - Melanoma
   • Neoplastic - Sarcoma

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Temperature of heat-induced retrieval	Steam for 40 min Steam for 20 min Steam for 8 min
   Analyte Extraction and Purification	Antigen retrieval	Enzymatic solution Trypsin Ficin Target retrieval system
   Biospecimen Preservation	Time in fixative	6 h 24 h 72 h
   Analyte Extraction and Purification	Decalcification solution/ duration	RBD Versenate S/P Decal

   View more