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Whole-specimen histopathology: a method to produce whole-mount breast serial sections for 3-D digital histopathology imaging.

Author(s): Clarke GM, Eidt S, Sun L, Mawdsley G, Zubovits JT, Yaffe MJ

Publication: Histopathology, 2007, Vol. 50, Page 232-42

PubMed ID: 17222252 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 gel and adhesive formulation used for the preservation of 3-D conformation of breast tissue specimens prior to slicing and fixation.

   Summary of Findings:

   Adhesion of the gel in which specimens were suspended to the actual specimen was temperature, formulation, and specimen size dependant. Overall rigidity, tissue adhesion supporting thin slicing, and compatibility with paraffin processing were best when specimens were suspended in 3.5% agar gel at 55 degrees C. Specimens suspended in100% HistoGel, 50% HistoMer, chitosan in acetic acid with potassium chloride, deacetylated chitosan chloride in 3.5% agar, albumin in 3.5% agar, carageenan, and agar (when larger specimens were used at lower suspension temperatures) were inadequate in terms of tissue adhesion. Specimens suspended in 5% gelatin and 1-1.5% gelatin in 2.5% agar became brittle and were incompatible with processing. Specimens in 5% albumin, 24% pectin, and chitosan in acetic acid without potassium chloride did not show adequate rigidity. The authors note that some drawbacks to the use of agar gel for preservation of 3-D conformation are that it does not adhere to skin which may be on the surface of a tissue specimen. The authors note that several dyes and pigments, as well as insertion of fibers into the gel failed to work for realignment purposes, but that the gel itself can be used as an external visible reference for aligning serial sections.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Morphology	Macroscopic observation

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Embedding medium	Agar Albumin Carageenan Chitosan Deacetylated chitosan chloride Gelatin HistoGel HistoMer Pectin
   Biospecimen Preservation	Fixative additive/buffer	Acetic acid Sodium chloride Potassium chloride
   Biospecimen Preservation	Embedding duration/condition	Room temperature 25 degrees C 35 degrees C 45 degrees C 55 degrees C

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2. Study Purpose

   The purpose of this study was to determine the effects of fixative, fixation duration, and processing schedule on immunohistochemistry and morphological preservation of whole-mount sectioned breast specimens. Routine specimens were fixed for 24 h in formalin and processed without gel suspension following a standard 14.5 hour processing schedule. For whole-mount sectioning, specimens were suspended in agar gel for preservation of 3-D conformation and sliced into 4-5 mm thick slices prior to experimental fixation and processing schedules.

   Summary of Findings:

   Fixation with 4% paraformaldehyde in 0.1 M Millonig's buffer yielded superior morphological preservation compared to fixation in 10% neutral buffered formalin, and a fixation time of 10-14 d was superior to 7 d. A processing schedule lasting 2 d was superior to ones lasting 4, 7, or 9 d, with the longer processing schedules resulting in difficult or impossible sectioning. Extended paraffin infiltration for 7 d maximized the yield of 4 uM thick serial sections compared to longer or shorter durations. The authors state the use of a vacuum oven was unable to reduce the time required for paraffin infiltration. IHC staining was comparable between routinely processed specimens and whole-mount processed tissue (fixed and processed using the optimum parameters mentioned above) for ER, PR, and Tab250 (Her2/Neu). The authors state that myosin smooth muscle IHC staining was also good but that E-cadherin and CB11 (Her2/Neu) staining were inadequate in whole-mount processed tissue.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin
   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Paraformaldehyde
   Biospecimen Preservation	Time in fixative	24 h 7 d 10-14 d
   Biospecimen Preservation	Duration of tissue/ specimen processing	14.5 h 2 d 4 d 7 d 9 d
   Biospecimen Preservation	Embedding duration/condition	With vacuum/pressure No vacuum/pressure 12 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d 11 d 12 d 13 d 14 d 15 d
   Immunohistochemistry Specific	Targeted peptide/protein	ER PR Myosin smooth muscle E-cadherin Her2/Neu
   Immunohistochemistry Specific	Type of antibody	Tab250 CB11
   Biospecimen Aliquots and Components	Type of slide	Whole-mount presentation Small section

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