Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Prolonging fixation time of an alternative fixative to formalin for dermatological samples using standard laboratory protocols.

Author(s): Smith J, Faria CSAA, Qvist CC, Melchior LC, Lauridsen T

Publication: J Clin Pathol, 2020, Vol. , Page

PubMed ID: 32669366 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared H&E, ABVG, IHC, and FISH staining scores as well as DNA integrity between FFPE and PFPE skin and tonsil specimens subjected to fixation for different durations. Skin from each of three mastectomy specimens and was divided into eight specimens of 3-5 mm and fixed within 4 h of surgery. Additionally, tonsil specimens that had been sectioned into 5 mm blocks, frozen in dry ice, and stored at -80°C were thawed at room temperature and fixed for this study. Specimens were fixed in PAXgene for 2 h, 24 h, 3 days, or 7 days at room temperature and then placed in stabilizer for 19-23 h. The remaining specimens were fixed in 10% neutral buffered formalin for 2 h, 24 h, 3 days, or 7 days at room temperature followed by an additional 19-23 h in formalin to enable processing alongside the PAXgene specimens. Specimens were paraffin-embedded using a HistoStar Embedding Workstation. Sections (1-1.5 µm) were hematoxylin and eosin and Alcian Blue van Gieson (ABVG) stained using a Tissue-Tek Prisma. H&E stained slides were scored 1-3 by two blinded laboratory scientists and one blinded pathologist based on nuclei, cytoplasm, other layers (connective tissue), and artifacts as well as intensity, color, and specificity of the staining and AVBG staining was evaluated by two blinded laboratory scientists based on the specificity and intensity of the staining. To investigate the effects in immunohistochemistry, sections were immunohistochemically stained using antibodies to CK5, CK7, CK-AE, MelanA, HMB45, S100, CD45, and MLH1 and evaluated by two blinded scientists based on signal intensity, sensitivity, and specificity. To investigate the effects on FISH, sections were stained using probes for FUS, SS18, and HER2 and staining was scored by a laboratory scientist as 0 (unsuitable), 1 (suitable for preliminary counting), 2 (sufficient for counting but with artifacts), or 3 (allows unambiguous analysis) based on signal and background. To evaluate the effects on DNA yield and integrity, DNA was extracted from four 10 µm sections that were stored at 4°C for up to 2 weeks using the Maxwell RSC DNA FFPE Kit. DNA yield and purity were assessed by spectrophotometry. DNA integrity was evaluated by PCR amplification of 100 bp, 200 bp, 300 bp, 400 bp, and 600 bp fragments and visualization using a QIAxcel Advanced system.

   Summary of Findings:

   Based on three observers, the FFPE specimen was found to have superior H&E staining to the matched PFPE specimen in 36 of 72 observations (50%), equivalent staining in 31 of 72 observations (43%), and the remaining five observations (7%) found better PFPE staining. The PFPE specimens also displayed more H&E scoring variation than the FFPE specimens (18.1% versus 12.6%). Importantly, the FFPE specimen was more likely to have superior rather than equivalent staining to the matched PFPE specimen with fixation of 2 h,  the FFPE was as likely to have equivalent as superior staining to the PFPE specimen in specimens fixed for ≥24 h, but the difference among fixation durations was not significant. While at most timepoints the FFPE specimen displayed superior FISH staining to the matched PFPE specimen, four specimens fixed for 24 h displayed equivalent staining and only two had superior staining in the FFPE specimen. The FISH staining intensity was lower in specimens stained for 2 h or 7 days but the observer was still able to score the specimens. ABVG staining intensity, contrast, and artifacts were equivalent in FFPE and PFPE specimens. Similarly, the FFPE and matched PFPE specimen were given the same IHC score in 68.3% of cases with FFPE scoring higher in 18% of cases and PFPE scoring higher in the remaining 15% of cases. Importantly, there were no significant effects on IHC scoring identified due to fixative or fixation duration and while the variation in scoring between the two observers varied among the antibodies, it was similar for FFPE and PFPE staining (10% versus 8.4%). DNA integrity was better in the PFPE specimen than the matched FFPE specimen in 62.5% of cases and was equivalent in the remaining 37.5% of cases but the difference in percentage low (1-2) versus high (3-4) was only significant for skin specimens. The best fixation duration for PFPE was 24 h to 3 days. Tonsil specimens tended to have more H&E artifacts, which may be attributable to the prior frozen storage.

   Biospecimens

   • Tissue - Skin
   • Tissue - Tonsil

   Preservative Types

   • Frozen
   • PAXgene
   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Other diagnoses
   • Neoplastic - Not specified

   Platform:

   Analyte	Technology Platform
   DNA	FISH
   DNA	PCR
   Morphology	H-and-E microscopy
   Protein	Immunohistochemistry
   Morphology	Light microscopy
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Tonsil Skin
   Immunohistochemistry Specific	Targeted peptide/protein	CK5 CK7 CK-AE MelanA HMB45 S100 CD45 MLH1
   Biospecimen Preservation	Time in fixative	2 h 24 h 3 days 7 days
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) PAXgene

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