A Critical Evaluation of the PAXgene Tissue Fixation System: Morphology, Immunohistochemistry, Molecular Biology, and Proteomics.
Author(s): Mathieson W, Marcon N, Antunes L, Ashford DA, Betsou F, Frasquilho SG, Kofanova OA, McKay SC, Pericleous S, Smith C, Unger KM, Zeller C, Thomas GA
Publication: Am J Clin Pathol, 2016, Vol. 146, Page 25-40
PubMed ID: 27402607 PubMed Review Paper? No
Purpose of Paper
This paper compared morphology, immunohistochemistry (IHC) staining, RNA and DNA yield and integrity, and RNA amplificability in matched PAXgene-fixed paraffin-embedded (PFPE) and formalin-fixed paraffin-embedded (FFPE) specimens. RNA and DNA yield and integrity and RNA amplificability using PFPE and FFPE specimens were compared to matched frozen specimens.
Conclusion of Paper
Although overall morphology was similar in FFPE and PFPE specimens, PFPE specimens had more cytoplasmic eosinophilia, resulting in better visualization but also shrinkage artifacts not noted in their FFPE counterparts. Optimization of the protocol allowed for comparable staining for most antibodies but significantly less staining was still observed in PFPE than FFPE specimens for three antibodies. RNA and DNA yield were lower from PFPE than FFPE specimens, but PFPE specimens had higher RNA integrity numbers (RIN), larger maximum RT-PCR amplicon size, lower cycle-threshold (CT) values, and more intact DNA as observed by electrophoresis. RINs were lower and CT values higher for RNA from PFPE specimens than frozen specimens, but the maximum amplicon size and DNA size by electrophoresis were comparable.
Studies
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Study Purpose
This study compared the IHC staining and morphology of matched FFPE and PFPE specimens. Morphology was investigated using 57 matched FFPE and PFPE specimens including colorectal, breast, lung, liver, renal, prostate, pancreas, myometrium, ovary, pelvic mass, and adrenal specimens. IHC was investigated using ten colorectal adenocarcinoma, nine matched colorectal normal adjacent specimens, nine lung carcinoma, and one lung aspergilloma. All specimens were divided and fixed in either 10% neutral buffered formalin for 24-48 h or in PAXgene for 1-4 h (most colon) or 8-24 h (all lung and one colon) and then paraffin-embedded. Tissue microarrays were constructed with selected areas of each specimen. Fresh 4 µm sections were dried for 30 min at 65˚C and H&E Safran stained on a automated Gemini tissue-stainer. Morphology and nuclear, cytoplasmic, and membrane details were assessed by a pathologist. IHC was performed using antibodies to thyroid transcription factor 1 (TTF1), p63, p40, Napsin A, cytokeratin (CK)5/6, CK7, CK20, Collagen IV, ki67, MutL homolog 1 (MLH1), MutS Homolog (MSH)2, MSH6, and PMS2 from multiple manufacturers. Antigen retrieval, pH, incubation time, antibody concentration, and detection kit were optimized for each antibody when necessary. Additionally, some antibodies required use of the UltraView Universal DAB detection kit. Every cell in each of three 400x fields was scored by a pathologist on a scale from 0 (no staining) to 4 (dark staining). The sum of the scores divided by 100 was the antibody/fixative score.
Summary of Findings:
Although overall morphology was comparable in FFPE and PFPE specimens, more eosinophilia was noted in the cytoplasm of PFPE lung and colon specimens than their FFPE counterparts. The authors found that the increased cytoplasmic eosinophilia increased contrast resulting in improved observation of cytoplasmic details. The authors also noted shrinkage artifacts in some PFPE specimens but not FFPE specimens.
Of the 28 antibodies used, seven (CK5/6, CK20, collagen IV and Ki67 from Dako and CK5/6, CK20, and p40 from Eurobio) produced comparable staining in FFPE and PFPE specimens, eight (p63, TTF1, CD56, and Napsin A from Ventana and p63, TTF1, CD56, and p40 from Diagnostic Biosystems) had weaker but still interpretable staining in PFPE than FFPE specimens, two (Collagen IV from Ventana and Napsin A from Eurobio) had weaker staining in FFPE than PFPE, four antibodies required protocol optimization, and the remaining eleven (CK7, MLH1, MSH2, MSH6, and PMS2 Ventana or Dako and Ki67 from Ventana) initially produced uninterpretable staining in PFPE specimens. Optimization of staining eliminated the difference in staining using antibodies to Napsin A, p63, p40, TTF1, CK7, Ki67, MSH2, MSH6, and MSH1, but staining of PFPE sections with antibodies to MLH1 and PMS2 from Ventana or MSH2 from Dako remained significantly less intense than that in FFPE specimens after optimization (P<0.05). The authors state that sub-optimal staining with CK7, CK20, and collagen IV from Dako or CK7 and collagen IV from Ventana was observed even after antigen retrieval.
Biospecimens
- Tissue - Colorectal
- Tissue - Breast
- Tissue - Liver
- Tissue - Lung
- Tissue - Adrenal Gland
- Tissue - Pancreas
- Tissue - Prostate
- Tissue - Ovary
- Tissue - Uterus
- Tissue - Kidney
Preservative Types
- PAXgene
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform Morphology H-and-E microscopy Protein Immunohistochemistry Morphology Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
PAXgene
Analyte Extraction and Purification Antigen retrieval pH 8.4 0 for 60 min
pH 6.0 for 60 min
pH 8.4 for 30 min
pH 6.0 for 36 min
pH 8.4 for 56 min
pH 6.0 for 32 min
pH 6.0 for 56 min
pH 6.0 for 8 min
Immunohistochemistry Specific Detection method Optiview DAB detection kit
Iview DAB detection kit
Immunohistochemistry Specific Antibody dilution/concentration 1:50
1:100
Manufacturer predilution
Analyte Extraction and Purification Incubation duration/condition 16 min
20 min
32 min
1 h
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Study Purpose
This study compared RNA and DNA yield and integrity and RNA amplicability in matched frozen FFPE and PFPE specimens and investigated if a demodification step could increase the maximum RT-PCR fragment size. A total of 37 tissues including both tumor (27) and normal adjacent tissues (6) (thirteen breast, nine liver, five renal, three prostate, two pancreas, one colon, one myometrium, one ovary, one pelvic mass, and one adrenal) were divided into three pieces. Specimens were fixed in 10% neutral buffered formalin for 24-48 h or in PAXgene for 1-8 h and then paraffin-embedded, or were snap-frozen in liquid nitrogen and stored at -80˚C. RNA and DNA were extracted from 20 µm FFPE sections after deparaffinization using xylene with the FFPE RNEasy kit and QIAamp DNA FFPE Tissue kit, respectively. RNA and DNA were extracted from 20 µm PFPE sections after deparaffinization using xylene with the PAXgene Tissue RNA and DNA kits. Frozen specimens were homogenized in a TissueLyser and RNA and DNA were extracted using the RNEasy mini kit and the QIAamp DNA FFPE Tissue kit, respectively. All RNA was treated with DNAse. RNA and DNA yield and purity were determined by spectrophotometry. RNA integrity was determined by bioanalyzer and DNA integrity was determined by electrophoresis. RNA was reverse transcribed directly or after a 1 h incubation at 70˚C and the 65, 265, 534, and 942 bp fragments of hydroxymethylbilane synthase (HMBS) were amplified by RT-PCR. Real-time PCR of actin, glial cell-derived neurotrophic factor (GDNF), GDNF family receptor alpha-1 (GFRa1), and Ret proto-oncogene (RET) was performed using RNA from a single liver tumor and an ovarian tumor.
Summary of Findings:
The mean RNA yield was significantly lower from PFPE than FFPE specimens (2.74 µg/section versus 0.71 µg/section, P<0.001), but the mean RIN was significantly higher (4.2 versus 2.3, P=4.8 x 10-8). However, the RIN was still significantly lower in PFPE than frozen specimens (4.2 versus 5.7, P<0.005). Interestingly, the largest range of RINs occurred in frozen blocks (2.2-8.3 for frozen and 2.2-6.7 for PFPE), which the authors attribute to variability of quality in clinical specimens. All four amplicons (65, 265, 534, and 942 bp) of HMBS were amplifiable in PFPE and frozen specimens but only the smallest amplicon (65 bp) was amplifiable in the FFPE specimens, regardless of inclusion of a demodification step (1 h at 70˚C). Real-time PCR cycle threshold (CT) values were highest for FFPE specimens, followed by PFPE and frozen specimens, but the differences were only significant for β-actin (P=0.02 for FFPE versus PFPE and P=0.01 for PFPE versus frozen).
DNA yield was lower from PFPE than FFPE specimens (2.95 µg/section versus 6.78 µg/section), but in contrast to results for RNA yield, the difference was not significant. Electrophoresis of DNA from PFPE and frozen specimens showed a band of >15 kbp, but DNA from FFPE specimens only had degraded DNA as evidence by a smear of <500 bp.
Biospecimens
- Tissue - Kidney
- Tissue - Breast
- Tissue - Adrenal Gland
- Tissue - Pancreas
- Tissue - Colorectal
- Tissue - Uterus
- Tissue - Lung
- Tissue - Liver
- Tissue - Prostate
Preservative Types
- Frozen
- Formalin
- PAXgene
Diagnoses:
- Neoplastic - Carcinoma
- Other diagnoses
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA RT-PCR RNA Spectrophotometry DNA Electrophoresis DNA Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
Formalin (buffered)
PAXgene
Analyte Extraction and Purification Incubation duration/condition 1 h at 70˚C
None
RT-PCR Specific Targeted nucleic acid HMBS
Real-time qRT-PCR Specific Targeted nucleic acid β-actin
GFRa1
GDNF
RET
RT-PCR Specific Length of gene fragment 65 bp
265 bp
534 bp
942 bp