Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system.

Author(s): Belloni B, Lambertini C, Nuciforo P, Phillips J, Bruening E, Wong S, Dummer R

Publication: J Clin Pathol, 2013, Vol. 66, Page 124-35

PubMed ID: 23125305 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 method of fixation on morphological preservation and IHC staining using 12 melanoma biopsy specimens from skin and lymph node metastases. A portion of each specimen was either embedded in optimal cutting temperature compound (OCT) and snap frozen in liquid nitrogen, fixed in formalin for 24 h at room temperature and embedded in normal paraffin, or fixed for 2-4 h at 4 degrees C in PAXgene fixative, transferred to PAXgene Tissue Stabilization Reagent for at least 24 h, and embedded in low melting paraffin. PAXgene paraffin blocks were stored at 4 degrees C in the dark while formalin blocks were stored at room temperature in the dark.

   Summary of Findings:

   There were no significant differences in morphological preservation between FFPE specimens and PAXPE specimens. Significantly weaker IHC staining was observed in PAXPE tissue than FFPE tissue for b-Raf, microphthalmia-associated transcription factor (MITF), cyclinD1 (CCND1), melanoma-associate antigen 4 (MAGEA4), and Tyrosinase, but protocols employed were specifically developed for FFPE tissue. No statistically significant differences in immunostaining intensity were seen for 6 other antigens. The authors report that a higher concentration of antibody improved Ki67 immunostaining in PAXPE which was initially insignificantly lower than that seen in FFPE tissue, but altering antibody concentration did not improve p53 immunostaining intensity in PAXPE (also insignificantly lower than FFPE), and altering antigen retrieval pH did not improve CCND1 or p53 immunostaining in PAXPE tissue. The addition of NP-40 to staining reactions was able to increase MITF, p53, and CCND1 immunostaining in PAXPE tissue.

   Biospecimens

   • Tissue - Skin
   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • Frozen
   • PAXgene

   Diagnoses:

   • Neoplastic - Melanoma

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) PAXgene Snap frozen
   Immunohistochemistry Specific	Targeted peptide/protein	MelanA Tyrosinase p16 MAGEA4 b-Raf CCND1 c-Kit Ki67 MITF p53 S100
   Immunohistochemistry Specific	Antibody dilution/concentration	1:100 1:200 1:500 1:1000
   Analyte Extraction and Purification	Antigen retrieval	pH 6 pH 9 No antigen retrieval
   Analyte Extraction and Purification	Cell/tissue permeabilization	NP-40 No NP-40

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2. Study Purpose

   The purpose of this study was to determine the effects of method of fixation on DNA and RNA analysis using 12 melanoma biopsy specimens from skin and lymph node metastases. A portion of each specimen was either embedded in OCT and snap frozen in liquid nitrogen, fixed in formalin for 24 h at room temperature and embedded in normal paraffin, or fixed for 2-4 h at 4 degrees C in PAXgene fixative, transferred to PAXgene Tissue Stabilization Reagent for at least 24 h, and embedded in low melting paraffin. PAXgene paraffin blocks were stored at 4 degrees C in the dark while formalin blocks were stored at room temperature in the dark. DNA was extracted from PAXgene and formalin-fixed tissue using the BiOstic Tissue DNA Isolation kit. RNA was extracted from PAXgene-fixed specimens using the PAXgene Tissue RNA kit, from formalin-fixed specimens using the BiOstic FFPE Tissue RNA Isolation Kit, and from snap frozen specimens using Trizol.

   Summary of Findings:

   There were no significant differences in DNA or RNA purity, as measured by spectrophotometry, between preservation method-specific extraction methods. Increasing fragment length decreased amplification success for both PAXPE and FFPE tissue in real time qRT-PCR and qPCR assays while the same was not true of frozen specimens (only tested in real time qPCR assays). This effect was more pronounced for DNA and RNA extracted from FFPE tissue than for that extracted from PAXPE tissue. While the largest Abelson murine leukemia viral oncogene homolog 1 (ABL1) mRNA fragment (806 bp) failed to amplify for either PAXPE or FFPE specimens, amplificability of the shorter two ABL1 fragments (114 bp and 398 bp) as well as Tyrosinase mRNA was generally better in PAXPE specimens than FFPE specimens. Real time qPCR amplification of BCL6 corepressor (BCOR) and calcium channel, voltage-dependent, gamma subunit 4 (CACNG4) was also generally more successful using DNA from PAXPE specimens than FFPE specimens. For about half of the specimens, the real time qPCR amplification quality of BCOR or CACNG4 was similar to or better for the PAXPE specimens than the frozen specimens.

   Biospecimens

   • Tissue - Skin
   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • Frozen
   • PAXgene

   Diagnoses:

   • Neoplastic - Melanoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	PCR
   DNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) PAXgene Snap frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	ABL1 kinase Tyrosinase
   Real-time qRT-PCR Specific	Length of gene fragment	114 bp 398 bp 806 bp 108 bp 338 bp 589 bp 712 bp
   Real-time qPCR Specific	Targeted nucleic acid	BCL6 CACNG4
   Real-time qPCR Specific	Length of gene fragment	145 bp 275 bp 553 bp 117 bp 277 bp 571 bp
   Analyte Extraction and Purification	Analyte isolation method	BiOstic Tissue DNA Isolation kit BiOstic FFPE Tissue RNA Isolation Kit PAXgene Tissue RNA kit Trizol

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