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Formaldehyde substitute fixatives: analysis of macroscopy, morphologic analysis, and immunohistochemical analysis.

Author(s): Moelans CB, Ter Hoeve N, van Ginkel JW, Ten Kate FJ, van Diest PJ

Publication: Am J Clin Pathol, 2011, Vol. 136, Page 548-56

PubMed ID: 21917676 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine the effects of fixative type, time in fixative and antigen retrieval on morphological preservation and immunohistochemical (IHC) staining of 17 different tissues.

Conclusion of Paper

Histological staining was more intense in tissues fixed with F-Solv and less intense in tissues fixed with RCL-2 or FineFIX compared to formalin-fixed specimens, but F-Solv led to insufficient staining for most histological stains. Formalin fixation was best for specimen integrity and tissue preservation followed by F-Solv and RCL-2 fixation. Fixation duration had only a limited effect on histological staining, but in specimens fixed for 2 months, structures were difficult to recognize, especially when fixed with F-Solv. IHC staining was best in formalin-fixed specimens followed by RCL-2-, FineFIX- and F-Solv-fixed specimens. The authors report that antigen retrieval steps recommended for formalin-fixed specimens often did not improve staining of specimens fixed with the alternative fixatives, and alternate treatments such as pepsin digestion were sometimes necessary.

Studies

  1. Study Purpose

    The purpose of this study was to determine the effects of fixative type and time in fixative on morphological preservation of 17 different tissue types.

    Summary of Findings:

    Histological staining was more intense in tissues fixed with F-Solv and less intense in tissues fixed with RCL-2 or FineFIX compared to formalin-fixed specimens. Formalin was best for specimen integrity and tissue preservation followed by F-Solv and RCL-2 fixation. Fixation with RCL-2 or FineFIX resulted in red blood cell lysis, loss of eosinophilic cytoplasmic granules, and tissue shrinkage, but nuclear details were best preserved in RCL-2 fixed specimens. Only formalin fixation allowed for structure recognition in lymph nodes. Fixation duration had a limited effect on staining, but in specimens fixed for 2 months, structures were difficult to recognize, especially in specimens fixed with F-Solv. The authors report the penetration speed for FineFIX and RCL-2 was similar to that of formalin, but that F-Solv did not adequately penetrate large specimens, even after fixation for 2 months. While formalin-, FineFIX- and RCL-2-fixed specimens yielded good results with most histological stains tested, F-Solv-fixed specimens were given insufficient or intermediate scores in most cases.

    Biospecimens
    Preservative Types
    • Formalin
    • Other Preservative
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    Morphology Light microscopy
    Morphology H-and-E microscopy
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation FineFIX
    Formalin (buffered)
    F-Solv
    RCL2
    Biospecimen Preservation Time in fixative 4 h
    24 h
    2-4 days
    1-2 weeks
    1-2 months
    Light microscopy Specific Type of tissue stain Periodic acid-Schiff
    Periodic acid-Schiff diastase
    Gordon-Sweet
    Jones silver stain
    Alcian blue
    Azan
    Elastic van-Gieson
    View more
  2. Study Purpose

    The purpose of this study was to determine the effects of fixative type, time in fixative and antigen retrieval method on IHC staining of 17 different tissue types.

    Summary of Findings:

    IHC staining was best in formalin-fixed specimens followed by RCL-2-, FineFIX- and F-Solv-fixed specimens. IHC staining of chromogranin A was good, regardless of fixative. IHC staining for estrogen receptor (ER) was inadequate in specimens fixed with the formalin alternatives. Staining for progesterone receptor (PR) and p63 was intermediate or lower in specimens fixed with the formalin alternatives. S-100 staining was suboptimal in the colonic nerve plexi fixed with the formalin alternatives, but optimal staining was achieved in F-Solv-fixed neurilemmoma. CD45, creatinine kinase, and cytokeratin AE1/3 staining were inadequate in F-Solv-fixed specimens, regardless of pretreatment. The authors report that pretreatment steps recommended for formalin-fixed specimens often did not improve staining of specimens fixed with the alternative fixatives, but alternate treatments such as pepsin digestion improved staining in some cases.

    Biospecimens
    Preservative Types
    • Formalin
    • Other Preservative
    Diagnoses:
    • Not specified
    Platform:
    AnalyteTechnology Platform
    Protein Immunohistochemistry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation F-Solv
    FineFIX
    Formalin (buffered)
    RCL2
    Biospecimen Preservation Time in fixative 4 h
    24 h
    2-4 days
    1-2 weeks
    1-2 months
    Immunohistochemistry Specific Targeted peptide/protein AE1/3
    Chromogranin A
    ER
    PR
    p63
    S-100
    CD45
    CAM5.2
    Vimentin
    Analyte Extraction and Purification Antigen retrieval Citrate
    Pepsin
    EDTA
    View more

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