TY - JOUR
T1 - Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?
AU - Frasquilho, Sonia G.
AU - Sanchez, Ignacio
AU - Yoo, Changyoung
AU - Antunes, Laurent
AU - Bellora, Camille
AU - Mathieson, William
N1 - Funding Information:
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The study was performed using internal funds of the Integrated Biobank of Luxembourg, provided by the Luxembourg Ministry of Higher Education and Research.
Publisher Copyright:
© The Author(s) 2021.
PY - 2021/6
Y1 - 2021/6
N2 - We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF−ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF−ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9–2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF−ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.
AB - We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF−ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF−ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9–2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF−ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.
KW - Illumina
KW - PFPE
KW - ScreenTape
KW - Xylene
KW - degradation
KW - degrade
KW - fixative
KW - formaldehyde
KW - integrity
KW - paraffin
UR - http://www.scopus.com/inward/record.url?scp=85106230658&partnerID=8YFLogxK
U2 - 10.1369/00221554211017859
DO - 10.1369/00221554211017859
M3 - Article
C2 - 34010071
AN - SCOPUS:85106230658
SN - 0022-1554
VL - 69
SP - 389
EP - 405
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 6
ER -